WO2025059062A1 - Genetic engineering of human hematopoietic stem/progenitor cells (hspcs) for locus-specific expression of therapeutic proteins - Google Patents

Abstract

The present disclosure provides compositions and methods comprising modified hematopoietic stem/progenitor cells (HSPCs) comprising an exogenous nucleic acid (e.g., a chimeric antigen receptor (CAR)) insertion into the endogenous CD33 or NKG2A locus.

Description

GENETIC ENGINEERING OF HUMAN HEMATOPOIETIC STEM/PROGENITOR CELLS (HSPCS) FOR LOCUS-SPECIFIC EXPRESSION OF THERAPEUTIC PROTEINS

CROSS-REFERENCE TO RELATED APPLICATION

The present application is entitled to priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/581,880 filed on September 11, 2023, which is herein incorporated by reference in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The Sequence Listing submitted herewith as an XML file named "046483- 7439USl.xml," created on September 10, 2024 and having a size of 436,526 bytes, is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Chimeric antigen receptor modified T (CAR-T) cells have received multiple FDA approvals for the treatment of hematologic malignancies. However, phase I clinical trials in solid tumors show partial responses at best, with limited CAR-T persistence. Correlative studies implicate the immunosuppressive tumor microenvironment (TME) as a major barrier to various forms of cancer immunotherapy. The TME consists of stromal and hematopoietic-derived cells that are recruited by malignant cells to support their growth and to restrain immune responses to tumor-associated antigens. Bone marrow derived myeloid cells such as tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) are key components of this suppressive milieu, and infdtration of epithelial cancers with M2 -like immunosuppressive macrophages is associated with adverse prognosis. However, despite interest in targeting myeloid cells in the TME, the therapeutic arsenal remains empty.

CAR-expressing monocyte-derived macrophages (CAR-M) have been developed as a therapeutic modality for adoptive cellular therapy in solid tumors. In solid tumor models CAR-M overcome intrinsic resistance mechanisms in the tumor microenvironment and reject tumors via direct phagocytosis and engagement of the adaptive immune system. This therapy recently received fast-track designation from the FDA and is now being tested clinically (NCT04660929). However, a major barrier to macrophage-based treatments is that, unlike T cells, macrophages do not form immunological memory and are not expected to persist long-term. Indeed, preliminary data have shown that, unlike T cells that remain detectable in the peripheral blood of leukemia patients for 10+ years, engineered macrophages persist in the blood for only hours after infusion.

Likewise, CAR-expressing NK cells have been tested in preclinical and early phase clinical trials for the treatment of both hematologic and solid organ malignancies. However, CAR-NK therapies show heterogenous responses and lack of persistence as they do not form classic immunologic memory, and to date there are no approved CAR-NK therapies. Thus, there is a need in the art for for more permanent strategies to durably modify innate immune cells, such as myeloid and NK cells, in order to achieve effective anti-cancer therapies. The present invention addresses this need.

SUMMARY

In one aspect, the present invention provides a modified hematopoietic stem/progenitor cell (HSPC) comprising a modification in the endogenous CD33 locus in which a nucleic acid has been inserted.

In another aspect, the present invention provides a modified hematopoietic stem/progenitor cell (HSPC) comprising a modification in the endogenous NKG2A locus in which an exogenous nucleic acid has been inserted.

In an embodiment, the exogenous nucleic acid inserted in the modified HSPC encodes a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain. In one embodiment, the antigen binding domain binds HER-2. In another embodiment, the antigen binding domain binds CD33. In another embodiment, the exogenous nucleic acid encodes IL-12. In one embodiment, the HSPC differentiates into an immune cell, such as a monocyte or macrophage. In another embodiment, the HSPC differentiates into a Natural Killer (NK) cell.

In another aspect, the present invention provides a method of generating a modified immune cell, where the method comprises introducing an exogenous nucleic acid into the CD33 locus of a hematopoietic stem/progenitor cell (HSPC) and allowing the HSPC to differentiate into an immune cell. In one embodiment, the exogenous nucleic acid is introduced via a CRISPR/Cas system. In a particular embodiment, the CRISPR/Cas system comprises Cas9, a guide RNA (gRNA) that targets the CD33 locus, and a template for homology-directed repair (HDRT) comprising a nucleotide sequence that encodes the exogenous nucleic acid. Tn one embodiment, the immune cell is a monocyte or macrophage.

In another aspect, the present invention provides a method of generating a modified immune cell comprising a chimeric antigen receptor (CAR), where the method comprises introducing an exogenous nucleic acid encoding the CAR into the CD33 locus of a hematopoietic stem/progenitor cell (HSPC) and allowing the HSPC to differentiate into an immune cell. In one embodiment, the exogenous nucleic acid is introduced via a CRISPR/Cas system. In a particular embodiment, the CRISPR/Cas system comprises Cas9, a guide RNA (gRNA) that targets the CD33 locus, and a template for homology-directed repair (HDRT) comprising a nucleotide sequence that encodes the exogenous CAR. In one embodiment, the immune cell is a monocyte or macrophage. In another embodiment, the immune cell is an NK cell.

In another aspect, the present invention provides a method of generating a modified immune cell in which an exogenous nucleic acid is introduced into the NKG2A locus of a hematopoietic stem/progenitor cell (HSPC) and the HSPC is allowed to differentiate into an immune cell. In one embodiment, the exogenous nucleic acid is introduced via a CRISPR/Cas system. In a particular embodiment, the CRISPR/Cas system comprises Cas9, a guide RNA (gRNA) that targets the NKG2A locus, and a template for homology-directed repair (HDRT) comprising a nucleotide sequence that encodes the exogenous nucleic acid.

In another aspect, the present invention provides a method of generating a modified immune cell comprising a chimeric antigen receptor (CAR) in which an exogenous nucleic acid encoding the CAR is introduced into the NKG2A locus of a hematopoietic stem/progenitor cell (HSPC) and the HSPC is allowed to differentiate into an immune cell. In one embodiment, the exogenous nucleic acid is introduced via a CRISPR/Cas system. In one embodiment, the CRISPR/Cas system comprises Cas9, a guide RNA (gRNA) that targets the NKG2A locus, and a template for homology-directed repair (HDRT) comprising a nucleotide sequence that encodes the exogenous CAR.

In another aspect, the present invention provides a pharmaceutical composition comprising the HSPC or population thereof of any of the exogenous nucleic acids (e.g., described herein, or the immune cell or population generated thereof. In another aspect, the present invention provides a method of treating a disease or disorder in a subject in need thereof, where a pharmaceutical composition in accordance with the present disclosure is administered to the subject. In one embodiment, the disease of disorder is cancer. In another embodiment, the immune cell or immune cell population in the pharmaceutical composition is capable of long-term persistence in vivo. In another embodiment, administration ofo the pharmaceutical composition alters the tumor microenvironment (TME).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

FIG. 1 : Model for engineered hematopoietic stem/progenitor cell (HSPC) treatment of human cancer patients. Left: Autologous HSPCs are collected from a patient with metastatic cancer and engineered with a tumor-specific CAR under an endogenous lineage-restricted promoter. The patient is then engrafted with gene-engineered stem cells after conditioning chemotherapy. Right: Engineered HSPCs differentiate in the bone marrow and express the CAR in a lineage-restricted fashion. CAR expressing cells are continuously produced and recruited into the TME, where they recognize the tumor-associated antigen and promote immune-mediated rejection.

FIGs 2A-2D: Tumor growth kinetics and immune cell recruitment to the TME are not altered by the absence of CD33. FIG. 2A: NSG mice engrafted with control (Ctrl) or CD33- CRISPR knockout (CD33ko) human HSPCs showed CD33ko persistence (left panel) and equivalent blood engraftment (right panel). FIG. 2B: Engrafted mice injected i.v. with 0.5xl0⁶ SKOV-3 tumor cells showed no difference in tumor burden by in vivo bioluminescence. The primary site of tumor growth was the lungs. FIGs. 2C-2D: SKOV-3 tumor bearing lungs showed equal recruitment of control and CD33ko macrophages by flow cytometry, defined as live/human CD45+, CD1 lb+/HLA-DR+ cells (FIG. 2C, top panel). Cells expressed the TAM marker CD206 (FIG. 2C, bottom panel). n.s.= not significant (p>0.05).

FIG. 3: Schema for knock-in of therapeutic cargo to the CD33 locus. A DNA doublestrand break is initiated at a lineage-specific targeted locus using CRISPR/Cas or other means and a template for homology-directed repair (HDRT) is provided either nonvirally (dsDNA, ssDNA) or virally (adeno-associated virus/AAV, integrase-deficient lentivirus/IDLV). The HDRT includes 5’ and 3’ homology arms (LHA/RHA) specific for the targeted gene. After undergoing HDR cells express the inserted construct specifically within the targeted cell lineage.

FIG. 4: Screening of gRNAs targeting CD33. M0LM14 cell lines were electroporated with no guide RNA, a control guide RNA targeting exon 2 of CD33 known to cause CD33 knockout, or a number of additional guide RNAs in both full-length and truncated forms. CD33 expression was assessed 14 days after electroporation by flow cytometry. Cell viability and CD33 knockout efficiency was most efficient for the truncated guide 4, which targets CD33 exon 1.

FIG. 5: Amplicon analysis of indels generated by guide 4. Genomic DNA was isolated from M0LM14 cells shown in FIG. 4 after 14 days and targeted amplicons of CD33 generated by PCR. Tracking indels by decomposition (TIDE) analysis showed indel frequency in cells targeted by either full-length or truncated Cas9 guide RNA/RNP complex using guide 4.

FIG. 6: HDR template design for knock-in of mCherry into exon 1 of CD33 targeted by guide RNA 4. MPLLLLLPLGSGGTSGVSKGEEDNMAII (SEQ ID NO: 351).

FIG. 7: CD33 engineering leads to persistent gene expression in vitro. Detection of knock-in to the CD33 locus. Primary human normal-donor (ND) CD34+ hematopoietic stem/progenitor cells (HSPCs) were electroporated with Cas9/ribonuclear protein (RNP) complex targeting either CD33 (CD33ko) or an irrelevant site (CD38ko) with non-viral dsDNA HDRT encoding for the fluorescent reporter protein mCherry. Targeted integration of mCherry could be detected by flow cytometry in HSPC with on-target RNP (CD33) but not irrelevant site (CD38ko) and was maintained >1 month during myeloid and macrophage differentiation.

FIG. 8: CD33 engineering leads to persistent gene expression in vitro. Genomic detection of on-target CD33 HDR knock-in HSPC. Primary normal donor CD34+ human HSPC were edited using Cas9 RNP containing either no guide RNA (NTC), CD33 guide RNA (33KO), or an off-target guide RNA (38KO) specific for the unrelated gene CD38. Indicated conditions were co-electroporated with a dsDNA HDRT encoding for mCherry (mC). Cells were cultured in vitro, and genomic DNA was isolated after 7 days, followed by PCR amplification using primers targeting the CD33 locus beyond the 3’ homology arm/RHA and the mCherry insert and agarose gel electrophoresis. Box indicates amplicon of expected size representing on-target insertion, which was confirmed by gel purification, Sanger sequencing of the PCR product, and alignment with the expected insertion sequence (right panel). Right panel, top sequence is SEQ ID NO: 347; and bottom sequence is SEQ ID NO: 348.

FIGs. 9A-9D: Engraftment of CD33 knock-in primary human HSPCs. Control (Ctrl), CD33 knockout (CD33ko), or CD33-knockout/mCherry knock-in (CD33mCherry) primary human normal donor (n.d.) CD34+ HSPCs were generated by Cas9/gRNA RNP electroporation +/- a dsDNA HDRT encoding mCherry. FIG. 9A: mCherry knock-in detected by flow cytometry after 4 days. FIG. 9B: NSG mice were engrafted with control, CD33ko, or CD33mCherry human HSPC after busulfan conditioning. mCherry expression was detected in peripheral blood monocytes for >16 weeks by flow cytometry. FIG. 9C: NSG mice engrafted with HSPC as in FIG. 9B were challenged at 8 weeks with SKOV3 cancer cells i.v. Tumor-bearing lung digests at harvest showed mCherry+ human immune infiltration of the TME by flow cytometry (gated on live/human CD45+/mouse CD45-). FIG. 9D: TME mCherry expression was restricted to HLA- DR+ APCs, which co-expressed the macrophage marker CD1 lb, but was not found in T (CD3+), B (CD19+) or NK (CD56+) cells. -30% of TME macrophages were mCherry+, reflecting -10% of the total human CD45+ infiltrate. FIG. 9A and FIG. 9B are representative data from one of 3 independent experiments with HSPC in each experiment derived from a different n.d. There were at least 4 engrafted mice per condition per experiment. FIG. 9C and FIG. 9D represent 1 n.d., with dots signifying individual engrafted mice.

FIGs. 10A-10B: The CD33 knock-in strategy is effective in all HSPC subsets including phenotypically immature hematopoietic stem cells. FIG. 10A: Gating strategy and FIG 10B: mCherry reporter detection within indicated human lineage-positive (Lin+) and Lin- cell populations in the bone marrow of NSG mice engrafted with control (Ctrl), CD33 knockout (CD33ko) or CD33 knock-in (CD33mCherry) human HPSCs by flow cytometry. Cells were detected 16 weeks post-engraftment by gating on human CD45+/murine CD45- on harvested single-cell suspensions from isolated bone marrow. Data is representative of 1 of 5 total mice per HSPC engraftment group.

FIGs. 11A-1 IB: CD33 engineered HSPCs maintain hematopoietic potency on secondary bone marrow engraftment. After 12 weeks, secondarily engrafted NSG mice were sacrificed and bone marrow harvested for flow cytometry. After gating on human CD45+/murine CD45- cells, mCherry was detected in both the total (top panel) and CD34+ (bottom panel) only in mice engrafted with CD33 knock-in cells. FIG. 12: Shows some embodiments of constructs used for CAR knock-into CD33 locus.

FIG. 13: Knock-in of potential therapeutic chimeric antigen receptors into the CD33 locus of primary HSPCs. CD33 knock-in primary human normal donor (n.d.) CD34⁺ HSPCs were generated by CD33 Cas9/gRNA RNP electroporation +/- a dsDNA HDRT encoding for a control HER2 CAR (Ctrl ki, not antibody detectable), anti-CD33 CAR (CAR33/containing a G4S scFv linker), anti-Her2 CAR (HER2CAR) containing a Myc Tag (5177 ki), or HER2CAR containing a G4S scFv linker (5178 ki) (SEQ ID NO: 367). 5 days after electroporation surface expression of CAR was detected by antibody staining for the G4S linker or Myc tag.

FIGs. 14A-14D: Stable genomic detection of on-target CD33 CAR knock-in. Primary normal donor CD34+ human HSPC were edited using a CD33 targeting Cas9 RNP with either mCherry (mC) or Her2CAR (5178) dsDNA HDRT. FIG. 14A: PCR amplification using a primer targeting the CD33 locus beyond the 5’ homology arm/LHA and a second primer specific for the Her2CAR insert for 5178 ki (or the mCherry insert for mC ki). Box indicates amplicon of expected size detected by agarose gel electrophoresis representing on-target insertion of 5178 Her2 CAR construct. FIG. 14B: On-target insertion was confirmed by gel purification, Sanger sequencing of the PCR product, and alignment with the expected insertion sequence (alignment top sequence: SEQ ID NO: 349; bottom sequence: SEQ ID NO: 350). FIG. 14C: Her2CAR knock in (5178 ki) CD34+ HSPCs were sorted for CAR positivity by fluorescence activated cell sorting (FACS) and differentiated into CD14+ macrophages for 60 days. CAR surface expression remained detectable by flow cytometry in -90% of sorted 5178 ki cells but was not detectable in mCherry knock-in (mC ki) macrophages. FIG. 14D: Genomic DNA was extracted from sorted macrophages after 60 days and amplified using an unbiased 3 -primer PCR reaction to generate amplicons that include both wild-type/indel CD33 alleles and the Her2 CAR knockin. Over 60% of sequences in 5178 ki macrophages showed on-target CAR insertion (HDR) by Inference of Crispr Edits (ICE) analysis. Sequencing of control amplicons generated using the same primers from mC ki macrophages showed only wild-type alleles or indels but no HDR knock-in of CAR. SEQ ID NOs: 410-425 are shown in FIG. 14D.

FIGs. 15A-15B: Expression and functional antitumor activity of CD33 knock-in HSPC- derived CAR macrophages. FIG. 15 A: Detection of in vitro differentiated macrophages expressing mCherry and Her2 CAR by flow cytometry. FIG. 15B: CD33 knockout/Her2 CAR knock-in cells were sorted by FACS and differentiated to macrophages for 21 days prior to incubation with the Her2+ cancer cell line SK0V3 expressing click beetle green luciferase for 48 hours. Normalized specific lysis was calculated as decline in luciferin fluorescence normalized to control unedited macrophages and compared to cells with knock-in of a non-HER2 specific CAR (irrelevant CAR).

FIGs. 16A-16D: Engraftment of CD33^HER2CAR^ primary human HSPCs in immunodeficient NSG mice. FIG. 16A shows a protocol for the detection of mCherry and Her2 CAR where quantification of the percentage of peripheral blood monocytes expressing mCherry and Her2 CAR (FIG. 16B-C) is determined by flow cytometry. Control (Ctrl), CD33 knockout (CD33ko), CD33-knockout/mCherry knock-in (CD33^mCherry), or CD33 knockout/HER2 CAR knock in (CD33^{HER2 CAR}) primary human normal donor (n.d.) CD34+ HSPCs were generated by Cas9/gRNA RNP electroporation +/- a dsDNA HDRT encoding mCherry or HER2 CAR. NSG mice were engrafted with control, CD33ko, CD33^mCherry, or human CD33^{HER2 CAR} human HSPC. Peripheral blood monocytes expressing mCherry and anti-Her2 CAR expression were detected in peripheral blood monocytes by flow cytometry (FIG. 16B-C) and a MethoCult assay was performed to determine the percentages of mCherry- and HER2 CAR-expressing cells within CFU-GM colonies (granulocyte/macrophage progenitors, identified by expression of CD 14) using flow cytometry(FIG. 16D).

FIGs. 17A-17D: Engraftment of CD33^HER2CAR^ myeloid cells lead to enhanced T cell infiltration in the TME. Single-cell RNA sequencing (scRNA seq) of tumors from NSGS mice challenged with the Her2+ cancer cell line SKOV3 after engraftment with CD33 engineered HSPCs was carried out to generate uniform manifold approximation and projection (UMAP) plots of CD33^mCherry and human CD33^{HER2 CAR} human HSPC cells colored by dataset and by cell types infiltrating the tumors after integration. The circled region indicates T cells.

FIGs. 18A-18E: Engraftment of CD33^HER2CAR^ myeloid cells in NSGS mice leads to enhanced CCL2 and IFNy-related chemokine expression. FIG. 18A shows a protocol for examining chemokine expression in peripheral blood monocytes following injection of NSG mice with Her2+ SKOV cells and engraftment with CD33^HER2CAR or CD33^mCherry CD34+ HSPCs. FIGs. 18B-18E show systemic expression levels of CCL2 (FIG. 18 A); CXCL9, CXCL10, and CXCL11 (FIG. 18 C); CCL17 and CCL20 (FIG. 18D); and CXCL5, CXCL1, and CXCL8 (FIG. 18E) in the peripheral blood plasma of mice engrafted with CD33^mCherry and CD33^HER2CAR myeloid cells. FIGs. 19A-19C: Enhanced CCL2/IFNy-r elated chemokine expression in CD33^HER2CAR engrafted NSGS mice appears to depend on CD3(j signaling. FIG. 19A shows a protocol for examining expression of CCL2 (FIG. 19B) or CXCL9 (FIG. 19C) in peripheral blood plasma following injection of NSGS mice with Her2+ SKOV cells and engraftment with CD33^mCherry, CD33^HER2CAR or CD33^HER2CARy CD34+ HSPCs.

FIGs. 20A-20D: Peripheral blood engraftment of myeloid cells in NSGS tumor bearingmice. FIG. 20A shows a protocol for examining the time course of immune cell engraftment, including monocyte/CD14 (FIG. 20B), T cell/CD3 (FIG. 20C), and B cell/CD19 (FIG. 20D) expression represented as the number of peripheral blood cells per pL expressing these markers as a function of time.

FIGs. 21A-21C: Peripheral blood engraftment mCherry and anti-HER2 CAR in NSGS mice. FIG. 21 A shows an experimental protocol for examining stable engraftment of mCherry and anti-HER2 C AR^ with engraftment, including % of peripheral blood monocytes expressing mCherry (FIG. 2 IB) and anti-HER2 CAR (FIG. 21C) as a function of time.

FIG. 22A-22C: Antitumor activity of anti-Her2 CAR knock-in HSC in vivo. FIG. 22A shows a protocol for examining antitumor activity of CD33^{HER2 CAR} CD34+ HSPCs in terms of tumor growth (FIG. 22B) and survival of NSGS mice as a function of time.

FIG. 23: Schema for knock-in of therapeutic cargo to the KLRCl locus. A DNA doublestrand break is initiated at a lineage-specific targeted locus using CRISPR/Cas or other means and a template for homology-directed repair (HDRT) is provided either nonvirally (dsDNA,ssDNA) or virally (adeno-associated virus/ AAV, integrase-deficient lentivirus/IDLV). The HDRT includes 5’ and 3’ homology arms (LHA/RHA) specific for the targeted gene. After undergoing HDR cells express the inserted construct specifically within the targeted cell lineage (NK cells). sgRNA: ACUGCAGAGAUGGAUAACAA (SEQ ID NO: 408).

FIGs. 24A-24B: EGFRt knock-in to NKG2A (KLRCl in primary human NK cells. Primary human normal donor Natural Killer (NK) cells were electroporated with Cas9/sgRNA RNP targeted KLRCl with/without a double-stranded DNA HDR template (HDRT) encoding a truncated non-signaling epidermal growth factor receptor (EGFRt) after culture for 16 hours with IL-12, 15, and 18 followed by maintenance and expansion with IL-2. FIG. 24A: Gating strategy for FIG. 24B flow cytometry plots showing successful tEGFR detection and NKG2A knockout after 7 days of in vitro culture in EGFR knock-in (KLRClEGFRt) but not mock electroporated (Ctrl) or NKG2A knockout-only (KLRC1 ko) controls.

FIG. 25: EGFRt knock-in to NKG2A/KLRC1 in primary human HSC followed by NK cell differentiation. Primary normal donor human HSPCs were electroporated with Cas9/sgRNA RNP targeting KLRC1 with/without a double-stranded DNAHDR template (HDRT) encoding a truncated non-signaling epidermal growth factor receptor (EGFRt) followed by culture for 18 days with StemSpam NK cell generation kit (StemCell Technologies). EGFRt knock-in was detected by flow cytometry in knock-in (KLRCl^EGFRt) but not mock electroporation control (Ctrl).

FIGs. 26A-26C: HDR template design for knock-in of CAR33 and tEGFR into exon 2 of NKG2A/KLRC1 is shown in FIGs. 26A-B, respectively. FIG. 26C shows additional details accompanying FIG. 26A (SEQ ID NOs: 426-431).

FIGs. 27A-27B: Knock-in of potential therapeutic chimeric antigen receptor against CD33 into the NKG2A locus of primary HSPCs. Stable genomic and flow cytometry detection of on-target KLRC1 CAR33 knock-in. Primary normal donor CD56+ human NK cells were edited using a KLRC1 targeting Cas9 RNP with CAR33 dsDNA through electroporation +/- a dsDNA HDRT encoding for an anti-CD33 CAR (CAR33/containing a G4S scFv linker). FIG. 27A: 7 days after electroporation surface expression of CAR was detected by antibody staining for the G4S linker. FIG. 27B: Sanger sequencing of the PCR amplification using a primer targeting the KLRC1 locus beyond the 5’ homology arm/LHA and a second primer specific for the CAR33 insert (EFl A promotor), and its alignment with the expected insertion sequence.

FIG. 28: NSG mice engrafted with control (Ctrl) or KLRC1-CRISPR knockout (NKG2Ako) human HSPCs showed NKG2Ako persistence (right) and equivalent blood engraftment and splenic infiltrations (left).

FIG. 29: Functional antitumor activity of NKG2A knock-in CAR33 NK cells. NKG2Aknockout/CAR33 knock-in, NKG2Aknockout/EGFRt knock-in, NKG2Aknockout or control NK cells were incubated with the CD33+ cancer cell line M0LM14 expressing click beetle green luciferase for 24 hours at different effector to target ratios (1 : 1, 1 :4 and 1 :8). Normalized specific lysis was calculated as decline in luciferin fluorescence normalized to control unedited macrophages and compared to cells with knock-in of a non-HER2 specific CAR (irrelevant CAR). DETAILED DESCRIPTION

Myeloid cells in the tumor microenvironment (TME) such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) express the lineage- restricted, pan-myeloid antigen CD33. Attempts to treat solid tumors by depleting these cells with CD33-directed bispecific T cell engagers or antibody drug conjugates have not been successful to date, perhaps in part because durable depletion of CD33-expressing MDSC also depletes CD33 -expressing progenitors, neutrophils and monocytes. Furthermore, these strategies do not have the potential to durably reprogram TAMs and MDSCs or to endow them with novel antitumor function. CRISPR-based deletion of CD33 in human hematopoietic stem/progenitor cells (HSPC) and the redundancy of CD33 for normal host hematopoiesis and myeloid cellular functions including engraftment, differentiation, cytokine production, signaling pathway activation, gene expression, phagocytosis, chemotaxis, and response to inflammatory stimuli have been demonstrated. CD33 deficiency is tolerated in humans, thus CD33 can be regarded as a myeloid-specific “safe harbor” locus for the expression of transgenes of interest. Herein, a novel CD33-based genetic engineering approach for myeloid immunotherapy of solid tumors is described. The overall concept is illustrated in FIG. 1.

CRISPR/Cas9-mediated non-homologous end joining (NHEJ) can be used to induce insertion/deletions (indels) at specific sites thus disrupting gene expression. In contrast, homology-directed repair (HDR) can be used to insert large DNA sequences at specific sites in the genome. The DNA template that encodes the transgene of interest (e.g. a CAR) can be delivered using viral or non-viral techniques in immune cells. Until the present study, insertion of a CAR cassette using a naked DNA template (i.e. not virally-encoded) had not been achieved in primary HSPCs. This is particularly important from the standpoint of translational feasibility, since use of viral vectors imposes significant challenges including the requirement for strict GMP manufacturing and testing. On the other hand, delivery of therapeutic gene cassettes using DNA oligodinucleotide (ODN) templates into human HSPC faces considerable challenges such as toxicity, viability, and possibly impairment of hematopoietic function. The work disclosed herein demonstrates that human HSPCs can be permanently modified to express a therapeutic transgene, using CRISPR/Cas9 technology to insert a CAR cassette into a validated myeloid locus. This thereby leads to continuous production of CAR-bearing myeloid cells that can disseminate widely and traffic to the TME upon their egress from the bone marrow. This is a novel approach to modification of the TME, utilizing genetically-modified HSPC transplantation and CAR-based immunotherapy.

It is to be understood that the methods described in this disclosure are not limited to particular methods and experimental conditions disclosed herein as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Furthermore, the experiments described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

A. Definitions

Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and/or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

That the disclosure may be more readily understood, select terms are defined below.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

“Activation,” as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions.

As used herein, to “alleviate” a disease means reducing the severity of one or more symptoms of the disease.

“Allogeneic” refers to a graft derived from a different animal of the same species.

“Xenogeneic” refers to a graft derived from an animal of a different species.

The term “antigen” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.

Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

As used herein, the term “autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

A “co- stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor.

A “co-stimulatory signal”, as used herein, refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to T cell proliferation and/or upregulation or downregulation of key molecules.

A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

The term “downregulation” as used herein refers to the decrease or elimination of gene expression of one or more genes.

“Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to an amount that when administered to a mammal, causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the invention. The immune response can be readily assessed by a plethora of art-recognized methods. The skilled artisan would understand that the amount of the composition administered herein varies and can be readily determined based on a number of factors such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like. “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

The term “epitope” as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response, inducing B and/or T cell responses. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. In general, an epitope is roughly about 10 amino acids and/or sugars in size. Preferably, the epitope is about 4- 18 amino acids, more preferably about 5-16 amino acids, and even more most preferably 6-14 amino acids, more preferably about 7-12, and most preferably about 8-10 amino acids. One skilled in the art understands that generally the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion of antigenic specificity and therefore distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope.

As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

The term “expand” as used herein refers to increasing in number, as in an increase in the number of cells. In one embodiment, the cells that are expanded ex vivo increase in number relative to the number originally present in the culture. In another embodiment, the cells that are expanded ex vivo increase in number relative to other cell types in the culture. The term "ex vivo," as used herein, refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e g., in a culture dish, test tube, or bioreactor). The term “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

“Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

“Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

The term “immune response” as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and/or activate lymphocytes to remove the antigen.

The term “immunosuppressive” is used herein to refer to reducing overall immune response.

“Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

The term “oligonucleotide” typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also includes an RNA sequence (i.e., A, U, C, G) in which “U” replaces “T ”

Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

“Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.

The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-beta, and/or reorganization of cytoskeletal structures, and the like.

A “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g., an aAPC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). A “subject” or “patient,” as used therein, may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is human.

A “target site” or “target sequence” refers to a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur. In some embodiments, a target sequence refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.

The term “therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

“Transplant” refers to a biocompatible lattice or a donor tissue, organ or cell, to be transplanted. An example of a transplant may include but is not limited to skin cells or tissue, bone marrow, and solid organs such as heart, pancreas, kidney, lung and liver. A transplant can also refer to any material that is to be administered to a host. For example, a transplant can refer to a nucleic acid or a protein.

The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

B. Methods of Generating Modified Immune Cells from Hematopoietic Stem/Progenitor Cell (HSPCs) The present disclosure provides methods for producing or generating modified immune cells or precursor thereofs (e.g., HSPC derived macrophages or NK cells) for adoptive immunotherapy.

In one aspect, the method comprises introducing an exogenous nucleic acid into the CD33 locus of a hematopoietic stem/progenitor cell (HSPC), and allowing the HSPC to differentiate into an immune cell (e.g., a monocyte or macrophage). In certain embodiments, the exogenous nucleic acid is introduced via a CRISPR/Cas system. In certain embodiments, the CRISPR/Cas system comprises Cas9, a guide RNA (gRNA) that targets the CD33 locus, and a template for homology-directed repair (HDRT) comprising a nucleotide sequence that encodes the exogenous nucleic acid.

In another aspect, the method comprises introducing an exogenous nucleic acid into the NKG2A locus of a hematopoietic stem/progenitor cell (HSPC), and allowing the HSPC to differentiate into an immune cell (e.g., an NK cell). In certain embodiments, the exogenous nucleic acid is introduced via a CRISPR/Cas system. In certain embodiments, the CRISPR/Cas system comprises Cas9, a guide RNA (gRNA) that targets the NKG2A locus, and a template for homology-directed repair (HDRT) comprising a nucleotide sequence that encodes the exogenous nucleic acid. In certain embodiments, the exogenous nucleic acid encodes IL- 12.

In certain embodiments, the exogenous nucleic acid encodes a molecule that alters the tumor microenvironment (TME). In certain embodiments, the exogenous nucleic acid encodes IL- 12.

Another aspect of the disclosure provides a method of generating a modified immune cell comprising a chimeric antigen receptor (CAR). The method comprises introducing an exogenous nucleic acid encoding the CAR into the CD33 locus of a hematopoietic stem/progenitor cell (HSPC), and allowing the HSPC to differentiate into an immune cell (e.g., a monocyte or macrophage). In certain embodiments, the exogenous nucleic acid is introduced via a CRISPR/Cas system. In certain embodiments, the CRISPR/Cas system comprises Cas9, a guide RNA (gRNA) that targets the CD33 locus, and a template for homology-directed repair (HDRT) comprising a nucleotide sequence that encodes the exogenous CAR.

Another aspect of the disclosure provides a method of generating a modified immune cell comprising a chimeric antigen receptor (CAR). The method comprises introducing an exogenous nucleic acid encoding the CAR into the NKG2A locus of a hematopoietic stem/progenitor cell (HSPC), and allowing the HSPC to differentiate into an immune cell (e g., a NK cell). Tn certain embodiments, the exogenous nucleic acid is introduced via a CRISPR/Cas system. In certain embodiments, the CRISPR/Cas system comprises Cas9, a guide RNA (gRNA) that targets the NKG2A locus, and a template for homology-directed repair (HDRT) comprising a nucleotide sequence that encodes the exogenous CAR.

The invention should be construed to include any chimeric antigen receptor known in the art or disclosed herein. Examples of CARs are described in U.S. Patent Nos.: 8,911,993, 8,906,682, 8,975,071, 8,916,381, 9,102,760, 9,101,584, 9,102,761, 9,777,061, and 11,197,919, all of which are incorporated herein by reference in their entireties. Amino acid sequences of exemplary CARs and CAR components for use in accordance with the present invention is shown in Table A:

Table A: Exemplary amino acid sequences.

In some embodiments, the CAR comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 370-390; or a variant thereof having one to five (e.g., 1, 2, 3, 4, or 5) amino acid modifications (e.g., substitutions).

In one embodiment, the antigen binding domain binds HER-2, where the antigen binding domain comprises the amino acid sequence of SEQ ID NO: 391 : DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLY SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGSTSG GGSGGGSGGGGSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY YCSRWGGDGFYAMDYWGQGTLVTVSS; or a variant thereof having one to five (e.g., 1, 2, 3, 4, or 5) amino acid modifications (e.g., substitutions).

In certain embodiments, the CAR comprises an antigen binding domain that binds CD33.

In one embodiment, the antigen binding domain binds CD33, where the antigen binding domain is encoded by the polynucleotide sequence of SEQ ID NO: 392: ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCT AGACCCGGATCCAACATCATGCTGACCCAGAGCCCTAGCAGCCTGGCCGTGTCTGCC GGCGAGAAAGTGACCATGAGCTGCAAGAGCAGCCAGAGCGTGTTCTTCAGCAGCTC CCAGAAGAACTACCTGGCCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAAGCTGC TGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGCAGC GGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCGTGCAGAGCGAGGACCTGGC CATCTACTACTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCGGAGGCACCAAGCT GGAAATCAAGAGAGGCGGCGGAGGCTCAGGCGGAGGCGGATCTAGTGGCGGAGGA TCTCAGGTGCAGCTGCAGCAGCCAGGCGCCGAGGTCGTGAAACCTGGCGCCTCTGT GAAGATGTCCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGAT CAAGCAGACCCCTGGACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAACG ACGACATCAGCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAG TCTAGCACCACCGCCTACATGCAGCTGTCCAGCCTGACCAGCGAGGACAGCGCCGT GTACTACTGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGGGAGCCGGCA CCACCGTGACCGTGTCATCT.

In another embodiment, the antigen binding domain binds CD33, where the antigen binding domain comprises the amino acid sequence of SEQ ID NO: 393: MALP VT ALLLPL ALLLHA ARPGSNIMLTQ SP S SL AVS AGEKVTMSCK S SQ S VFF S S SQK NYLAWYQQIPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQSEDLAIYYCHQ YLSSRTFGGGTKLEIKRGGGGSGGGGSSGGGSQVQLQQPGAEVVKPGASVKMSCKASG YTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFKGKATLTADKSSTTAYMQLSS LTSEDSAVYYCAREVRLRYFDVWGAGTTVTVSS; or a variant thereof having one to five (e.g., 1, 2, 3, 4, or 5) amino acid modifications (e.g, substitutions).

In some embodiments, the CAR comprises an antigen binding domain that binds CD33, wherein the CAR is encoded by the polynucleotide sequence of SEQ ID NO: 394: ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGCCGCT AGACCCGGATCCAACATCATGCTGACCCAGAGCCCTAGCAGCCTGGCCGTGTCTGCC GGCGAGAAAGTGACCATGAGCTGCAAGAGCAGCCAGAGCGTGTTCTTCAGCAGCTC CCAGAAGAACTACCTGGCCTGGTATCAGCAGATCCCCGGCCAGAGCCCCAAGCTGC TGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGCAGC GGCTCTGGCACCGACTTCACCCTGACAATCAGCAGCGTGCAGAGCGAGGACCTGGC CATCTACTACTGCCACCAGTACCTGAGCAGCCGGACCTTTGGCGGAGGCACCAAGCT GGAAATCAAGAGAGGCGGCGGAGGCTCAGGCGGAGGCGGATCTAGTGGCGGAGGA TCTCAGGTGCAGCTGCAGCAGCCAGGCGCCGAGGTCGTGAAACCTGGCGCCTCTGT GAAGATGTCCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATCCACTGGAT CAAGCAGACCCCTGGACAGGGCCTGGAATGGGTGGGAGTGATCTACCCCGGCAACG ACGACATCAGCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAG TCTAGCACCACCGCCTACATGCAGCTGTCCAGCCTGACCAGCGAGGACAGCGCCGT GTACTACTGCGCCAGAGAAGTGCGGCTGCGGTACTTCGATGTGTGGGGAGCCGGCA CCACCGTGACCGTGTCATCTTCCGGAGAGAGCAAGTACGGCCCTCCCTGCCCCCCT TGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCC AAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGT GTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC ACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTG TCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAA GGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGG GCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACC AAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCC GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGT GCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCC GGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAAC CACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATGATCTACATCTGGGC

GCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAG TACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAA GGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAA GCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACG ATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAG GAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAG GCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATG GCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC

AGGCCCTGCCCCCTCGC

In other embodiments, the CAR comprises an antigen binding domain that binds CD33, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 395:

MALP VT ALLLPL ALLLHAARPGSNIMLTQ SP S SL AVS AGEK VTMSCK S SQ S VFF S S SQK NYLAWYQQIPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQSEDLAIYYCHQ YLSSRTFGGGTKLEIKRGGGGSGGGGSSGGGSQVQLQQPGAEVVKPGASVKMSCKASG YTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFKGKATLTADKSSTTAYMQLSS LTSEDSAVYYCAREVRLRYFDVWGAGTTVTVSSSGESKYGPPCPPCPAPEFLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYR VVS VLTVLHQDWLNGKEYKCKVSNKGLP S SIEKTISK AKGQPREPQ VYTLPP SQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGKMIYIWAPLAGTCGVLLLSLVITLYCKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNE LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR; or a variant thereof having one to five (e.g., 1, 2, 3, 4, or 5) amino acid modifications (e.g., substitutions).

In various embodiments, the CARs described herein can include a spacer region located between the binding domain (e.g., a HER2 or CD33 scFv) and the transmembrane domain. A variety of different spacers can be used e.g., spacers comprising at least a portion of a human Fc region, for example a hinge portion of a human Fc region or a CH3 domain or variants thereof.

In some embodiments, the spacer region located between the binding domain (e.g., a HERZ or CD33 scFv) and the transmembrane domain comprises the amino acid sequence of: AAA; GGGSSGGGSG (SEQ ID NO: 396); ESKYGPPCPPCP (SEQ ID NO: 397) (IgG4 hinge); lEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 398) (CD28 hinge); or TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 399) (CD8 hinge).

In other embodiments, the CARs described herein comprise a transmembrane domain. In some embodiments, the transmembrane domain comprises the amino acid sequence of: LCYLLDGILFIYGVILTALFL (SEQ ID NO: 400) (CD3z transmembrane domain); FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 401) or MFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 409) (CD28 transmembrane domain);

MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 402) (CD4 transmembrane domain); or IISFFLALTSTALLFLLFFLTLRFSVV (SEQ ID NO: 403) (4-1BB transmembrane domain).

In other embodiments, the CARs described herein comprise one or more (e.g., two) costimulatory domains. In some embodiments, the costimulatory domain(s) are located between the transmembrane domain and the CD3z signaling domain.

Nonlimiting examples of suitable costimulatory domains together with the sequence of the CD3z signaling domain include: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 404) (CD3z aignaling domain); RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 405) (CD28 costimulatory domain);

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 406) (4-1BB costimulatory domain); and

ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 407) (0X40 costimulatory domain).

In certain embodiments, the immune cell is a monocyte or macrophage. In certain embodiments, the immune cell is a T cell. In certain embodiments, the immune cell is a Natural Killer (NK) cell.

In certain embodiments, the modified HSPCs are differentiated into immune cells in vivo. In certain embodiments, the modified HSPCs are differentiated ex vivo. In certain embodiments, the modified HSPCs are differentiated in vitro.

In certain embodiments, the exogenous nucleic acid is introduced into the HSPC via a CRISPR/Cas system. In certain embodiments, the CRISPR/Cas system comprises Cas9, at least one guide RNA (gRNA), and template for homology-directed repair (HDRT). In certain embodiments, the gRNA targets the CD33 locus. In certain embodiments, the gRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1-144. In certain embodiments, the gRNA targets the NKG2A locus.

In certain embodiments, the HDRT is provided nonvirally (e.g., dsDNA, ssDNA). In certain embodiments, the HDRT is provided virally (e.g., adeno-associated virus/ AAV, integrase-deficient lentivirus/IDLV).

The CRISPR/Cas9 system is a facile and efficient system for inducing targeted genetic alterations. Target recognition by the Cas9 protein requires a ‘seed’ sequence within the guide RNA (gRNA) and a conserved di-nucleotide containing protospacer adjacent motif (PAM) sequence upstream of the gRNA-binding region. The CRISPR/Cas9 system can thereby be engineered to cleave virtually any DNA sequence by designing the gRNA to target a particular sequence. The Cas9 protein and guide RNA form a complex that identifies and cleaves target sequences. Cas9 is comprised of six domains: REC I, REC II, Bridge Helix, PAM interacting, HNH, and RuvC. The REC I domain binds the guide RNA, while the Bridge helix binds to target DNA. The HNH and RuvC domains are nuclease domains. Guide RNA is engineered to have a 5’ end that is complementary to the target DNA sequence. Upon binding of the guide RNA to the Cas9 protein, a conformational change occurs activating the protein. Once activated, Cas9 searches for target DNA by binding to sequences that match its protospacer adjacent motif (PAM) sequence. A PAM is a two or three nucleotide base sequence within one nucleotide downstream of the region complementary to the guide RNA. In one non-limiting example, the PAM sequence is 5’-NGG-3’. When the Cas9 protein finds its target sequence with the appropriate PAM, it melts the bases upstream of the PAM and pairs them with the complementary region on the guide RNA. Then the RuvC and HNH nuclease domains cut the target DNA after the third nucleotide base upstream of the PAM.

CRISPR/Cas gene disruption occurs when a guide nucleic acid sequence specific for a target gene and a Cas endonuclease are introduced into a cell and form a complex that enables the Cas endonuclease to introduce a double strand break at the target gene. In certain embodiments, the CRISPR/Cas system comprises an expression vector, such as, but not limited to, a pAd5F35-CRISPR vector. In other embodiments, the Cas expression vector induces expression of Cas9 endonuclease. Other endonucleases may also be used, including but not limited to, Casl2a (Cpfl), T7, Cas3, Cas8a, Cas8b, CaslOd, Csel, Csyl, Csn2, Cas4, CaslO, Csm2, Cmr5, Fokl, other nucleases known in the art, and any combinations thereof.

In certain embodiments, inducing the Cas expression vector comprises exposing the cell to an agent that activates an inducible promoter in the Cas expression vector. In such embodiments, the Cas expression vector includes an inducible promoter, such as one that is inducible by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, for example doxycycline). Other inducible promoters known by those of skill in the art can also be used. The inducing agent can be a selective condition (e.g., exposure to an agent, for example an antibiotic) that results in induction of the inducible promoter. This results in expression of the Cas expression vector.

As used herein, the term “guide RNA” or “gRNA” refer to any nucleic acid that promotes the specific association (or “targeting”) of an RNA-guided nuclease such as a Cas9 to a target sequence (e.g., a genomic or episomal sequence) in a cell.

As used herein, a “modular” or “dual RNA” guide comprises more than one, and typically two, separate RNA molecules, such as a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), which are usually associated with one another, for example by duplexing. gRNAs and their component parts are described throughout the literature (see, e.g., Briner et al. Mol. Cell, 56(2), 333-339 (2014), which is incorporated by reference).

As used herein, a “unimolecular gRNA,” “chimeric gRNA,” or “single guide RNA (sgRNA)” comprises a single RNA molecule. The sgRNA may be a crRNA and tracrRNA linked together. For example, the 3’ end of the crRNA may be linked to the 5’ end of the tracrRNA. A crRNA and a tracrRNA may be joined into a single unimolecular or chimeric gRNA, for example, by means of a four nucleotide (e.g., GAAA) “tetraloop” or “linker” sequence bridging complementary regions of the crRNA (at its 3' end) and the tracrRNA (at its 5' end).

As used herein, a “repeat” sequence or region is a nucleotide sequence at or near the 3’ end of the crRNA which is complementary to an anti-repeat sequence of a tracrRNA.

As used herein, an “anti-repeat” sequence or region is a nucleotide sequence at or near the 5’ end of the tracrRNA which is complementary to the repeat sequence of a crRNA.

Additional details regarding guide RNA structure and function, including the gRNA / Cas9 complex for genome editing may be found in, at least, Mali et al. Science, 339(6121), 823- 826 (2013); Jiang et al. Nat. Biotechnol. 31(3). 233-239 (2013); and Jinek et al. Science, 337(6096), 816-821 (2012); which are incorporated by reference herein.

As used herein, a “guide sequence” or “targeting sequence” refers to the nucleotide sequence of a gRNA, whether unimolecular or modular, that is fully or partially complementary to a target domain or target polynucleotide within a DNA sequence in the genome of a cell where editing is desired. Guide sequences are typically 10-30 nucleotides in length, preferably 16-24 nucleotides in length (for example, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides in length), and are at or near the 5' terminus of a Cas9 gRNA.

As used herein, a “target domain” or “target polynucleotide sequence” or “target sequence” is the DNA sequence in a genome of a cell that is complementary to the guide sequence of the gRNA.

In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guide sequence is designed to have some complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In certain embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In other embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or nucleus. Typically, in the context of a CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) the target sequence. As with the target sequence, it is believed that complete complementarity is not needed, provided this is sufficient to be functional.

In certain embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell, such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas nuclease, a crRNA, and a tracrRNA could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5’ with respect to (“upstream” of) or 3’ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In certain embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron).

In certain embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme). A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in U.S. Patent Appl. Publ. No. US20110059502, incorporated herein by reference. In certain embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence.

Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian and non-mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CRISPR system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell (Anderson, 1992, Science 256:808-813; and Yu, et al., 1994, Gene Therapy 1 : 13-26).

In some embodiments, the CRISPR/Cas is derived from a type II CRISPR/Cas system. In other embodiments, the CRISPR/Cas sytem is derived from a Cas9 nuclease. Exemplary Cas9 nucleases that may be used in the present invention include, but are not limited to, S. pyogenes Cas9 (SpCas9), S. aureus Cas9 (SaCas9), S. thermophilus Cas9 (StCas9), N meningitidis Cas9 (NmCas9), C. jejuni Cas9 (CjCas9), and Geobacillus Cas9 (GeoCas9).

In general, Cas proteins comprise at least one RNA recognition and/or RNA binding domain. RNA recognition and/or RNA binding domains interact with the guiding RNA. Cas proteins can also comprise nuclease domains (i. e. , DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, as well as other domains. The Cas proteins can be modified to increase nucleic acid binding affinity and/or specificity, alter an enzymatic activity, and/or change another property of the protein. In certain embodiments, the Cas-like protein of the fusion protein can be derived from a wild type Cas9 protein or fragment thereof. In other embodiments, the Cas can be derived from modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein can be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, and so forth) of the protein. Alternatively, domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild type Cas9 protein. In general, a Cas9 protein comprises at least two nuclease (i.e., DNase) domains. For example, a Cas9 protein can comprise a RuvC-like nuclease domain and a HNH-like nuclease domain. The RuvC and HNH domains work together to cut single strands to make a double-stranded break in DNA. (Jinek, et al., 2012, Science, 337:816-821). In certain embodiments, the Cas9-derived protein can be modified to contain only one functional nuclease domain (either a RuvC-like or a HNH-like nuclease domain). For example, the Cas9-derived protein can be modified such that one of the nuclease domains is deleted or mutated such that it is no longer functional (i.e., the nuclease activity is absent). In some embodiments in which one of the nuclease domains is inactive, the Cas9-derived protein is able to introduce a nick into a double-stranded nucleic acid (such protein is termed a “nickase”), but not cleave the doublestranded DNA. In any of the above-described embodiments, any or all of the nuclease domains can be inactivated by one or more deletion mutations, insertion mutations, and/or substitution mutations using well-known methods, such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, as well as other methods known in the art.

In one non-limiting embodiment, a vector drives the expression of the CRISPR system. The art is replete with suitable vectors that are useful in the present invention. The vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. The vectors of the present invention may also be used for nucleic acid standard gene delivery protocols. Methods for gene delivery are known in the art (U.S. Patent Nos. 5,399,346, 5,580,859 & 5,589,466, incorporated by reference herein in their entireties).

Further, the vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (4th Edition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2012), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sindbis virus, gammaretrovirus and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01/96584; WO 01/29058; and U.S. Patent No. 6,326,193). In some embodiments, guide RNA(s) and Cas9 can be delivered to a cell as a ribonucleoprotein (RNP) complex (e.g., a Cas9/RNA-protein complex). RNPs are comprised of purified Cas9 protein complexed with gRNA and are well known in the art to be efficiently delivered to multiple types of cells, including but not limited to stem cells and immune cells (Addgene, Cambridge, MA, Minis Bio LLC, Madison, WI). In some embodiments, the Cas9/RNA-protein complex is delivered into a cell by electroporation.

The HSPC cells of the present inventon can be edited by any gene editing technology known to those skilled in the art. Gene editing technologies include, without limitation, homing endonucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector (TALE) nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)- associated protein 9 (Cas9). Homing endonucleases generally cleave their DNA substrates as dimers, and do not have distinct binding and cleavage domains. ZFNs recognize target sites that consist of two zinc-finger binding sites that flank a 5- to 7-base pair (bp) spacer sequence recognized by the FokI cleavage domain. TALENs recognize target sites that consist of two TALE DNA-binding sites that flank a 12- to 20-bp spacer sequence recognized by the FokI cleavage domain. Accordingly, one of skill in the art would be able to select the appropriate gene editing technology for the present invention.

C. Modified Hematopoietic Stem/Progenitor Cells (HSPCs)

Provided herein are modified hematopoietic stem/progenitor cells (HSPCs) for use in immunotherapy. In one aspect, the disclosure provides a modified hematopoietic stem/progenitor cell (HSPC) comprising a modification in the endogenous CD33 locus, wherein an exogenous nucleic acid has been inserted into the CD33 locus. In certain embodiments, the exogenous nucleic acid encodes a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain. In certain embodiments, the antigen binding domain of the CAR binds CD33 or HER-2.

In certain embodiments, the exogenous nucleic acid encodes a molecule that alters the tumor microenvironment (TME). In certain embodiments, the exogenous nucleic acid encodes IL-12.

In certain embodiments, the HSPC differentiates into an immune cell. In certain embodiments, the immune cell is a monocyte or macrophage. Another aspect of the disclosure provides a modified hematopoietic stem/progenitor cell (HSPC) comprising a modification in the endogenous NKG2A locus, wherein an exogenous nucleic acid has been inserted into the NKG2A locus. In certain embodiments, the exogenous nucleic acid encodes a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain. In certain embodiments, the antigen binding domain of the CAR binds HER-2.

In certain embodiments, the exogenous nucleic acid encodes a molecule that alters the tumor microenvironment (TME). In certain embodiments, the exogenous nucleic acid encodes IL- 12.

In certain embodiments, the HSPC differentiates into an immune cell. In certain embodiments, the immune cell is a Natural Killer (NK) cell.

In some aspects, the disclosure provides populations of modified HSPCs. In certain embodiments, the HSPC populations include those in which at least or greater than about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of HSPCs cells contain the desired genetic modification. For example, about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of HSPCs in a population of cells into which an agent (e.g. gRNA/Cas9) for knock-in or genetic disruption of endogenous gene was introduced contain the genetic disruption.

In some aspects, the disclosure provides populations of immune cells that have been differentiated from the modified HSPCs contemplated herein (e.g., HSPCs in which an exogenous nucleic acid has been inserted into the CD33 or NKG2A locus). In some embodiments, the immune cells in the composition retain a phenotype of the immune cell or cells compared to the phenotype of cells in a corresponding or reference composition when assessed under the same conditions. In some embodiments, cells in the composition include naive cells, effector memory cells, central memory cells, stem central memory cells, effector memory cells, and long-lived effector memory cells. In some embodiments, the percentage of immune cells expressing the exogenous nucleic acid (e.g., CAR) exhibit a long-lived, persistent phenotype. In some embodiments, such property, activity or phenotype can be measured in an in vitro assay, such as by incubation of the cells in the presence of an antigen targeted by the CAR.

As used herein, reference to a "corresponding composition" or a "corresponding population of immune cells" (also called a "reference composition" or a "reference population of cells") refers to immune cells obtained, isolated, generated, produced and/or incubated under the same or substantially the same conditions, except that the immune cells or population of immune cells were not introduced with the agent. In some aspects, except for not containing introduction of the agent, such immune cells are treated identically or substantially identically as immune cells that have been introduced with the agent, such that any one or more conditions that can influence the activity or properties of the cell, including the upregulation or expression of the inhibitory molecule, is not varied or not substantially varied between the cells other than the introduction of the agent.

Methods and techniques for assessing the expression and/or levels of T cell markers are known in the art. Antibodies and reagents for detection of such markers are well known in the art, and readily available. Assays and methods for detecting such markers include, but are not limited to, flow cytometry, including intracellular flow cytometry, ELISA, ELISPOT, cytometric bead array or other multiplex methods, Western Blot and other immunoaffinity-based methods. In some embodiments, antigen receptor (e.g. TCR and/or CAR)-expressing cells can be detected by flow cytometry or other immunoaffinity based method for expression of a marker unique to such cells, and then such cells can be co-stained for another T cell surface marker or markers.

As used herein, the term "introducing" encompasses a variety of methods of introducing DNA into a cell, either in vitro or in vivo, such methods including transformation, transduction, transfection (e.g. electroporation), and infection. Vectors are useful for introducing DNA encoding molecules into cells. Possible vectors include plasmid vectors and viral vectors. Viral vectors include retroviral vectors, lentiviral vectors, or other vectors such as adenoviral vectors or adeno-associated vectors.

Thus, provided are cells (e.g. modified HSPCs), compositions, and methods that enhance function in adoptive cell therapy, including those offering improved efficacy, such as by increasing activity and potency of administered genetically engineered cells, while maintaining persistence or exposure to the transferred cells over time. In some embodiments, the genetically engineered cells, exhibit increased expansion and/or persistence when administered in vivo to a subject, as compared to certain available methods. In some embodiments, the provided cells exhibit increased persistence when administered in vivo to a subject. In some embodiments, the persistence of genetically engineered immune cells, in the subject upon administration is greater as compared to that which would be achieved by alternative methods, such as those involving administration of cells genetically engineered by methods in which cells were not modified. In some embodiments, the persistence is increased at least or about at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more.

In some embodiments, the degree or extent of persistence of administered cells can be detected or quantified after administration to a subject. For example, in some aspects, quantitative PCR (qPCR) is used to assess the quantity of cells expressing the exogenous nucleic acid (e.g., CAR) in the blood or serum or organ or tissue (e.g., disease site) of the subject. In some aspects, persistence is quantified as copies of DNA or plasmid encoding the exogenous receptor per microgram of DNA, or as the number of receptor-expressing cells per microliter of the sample, e.g., of blood or serum, or per total number of peripheral blood mononuclear cells (PBMCs) or white blood cells per microliter of the sample. In some embodiments, flow cytometric assays detecting cells expressing the receptor generally using antibodies specific for the receptors also can be performed. Cell-based assays may also be used to detect the number or percentage of functional cells, such as cells capable of binding to and/or neutralizing and/or inducing responses, e.g., cytotoxic responses, against cells of the disease or condition or expressing the antigen recognized by the receptor. In any of such embodiments, the extent or level of expression of another marker associated with the exogenous nucleic acid (e.g. CAR) can be used to distinguish the administered cells from endogenous cells in a subject.

D. Methods of Treatment

Also provided herein are methods of treating a disease or disorder (e.g., cancer) in a subject in need thereof with cell-based immunotherapies comprising CD33 knock-in HSPCs comprising a CAR, CD33 knock-in HSPC-derived immune cells comprising a CAR (e.g., CAR macrophages), and NKG2A knock-in HSPCs comprising a CAR, NKG2A knock-in HSPC- derived immune cells comprising a CAR (e.g., CAR NK cells).

In one aspect, the disclosure provides a method of treating a disease or disorder (e.g., cancer) in a subject in need thereof. The method comprises administering to the subject, a modified hematopoietic stem/progenitor cell (HSPC) comprising a modification in the endogenous CD33 locus, wherein an exogenous nucleic acid has been inserted into the CD33 locus. In certain embodiments, the exogenous nucleic acid encodes a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain. In certain embodiments, the antigen binding domain binds CD33 or HER-2.

In certain embodiments, the exogenous nucleic acid encodes IL-12. In certain embodiments, the HSPC differentiates into an immune cell (e.g., a monocyte or macrophage).

In another aspect, the disclosure provides a method of treating a disease or disorder (e.g., cancer) in a subject in need thereof. The method comprises administering to the subject, a modified hematopoietic stem/progenitor cell (HSPC) comprising a modification in the endogenous NKG2A locus, wherein an exogenous nucleic acid has been inserted into the NKG2A locus. In certain embodiments, the exogenous nucleic acid encodes a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain. In certain embodiments, the antigen binding domain binds CD33 or HER-2.

In certain embodiments, the exogenous nucleic acid encodes IL- 12. In certain embodiments, the HSPC differentiates into an immune cell (e.g., a NK cell).

The CD33 or NKG2A knock-in HSPCs or HSPC-derived immune cells (e.g., CAR macrophages, CAR NK cells) described herein may be included in a composition for immunotherapy. The composition may include a pharmaceutical composition and further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified immune cells may be administered.

Methods for administration of immune cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8( 10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338. In some embodiments, the cell therapy, e.g., adoptive cell therapy is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject. In some embodiments, the cell therapy, e.g., adoptive cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

In some embodiments, the subject has been treated with a therapeutic agent targeting the disease or condition, e.g. the tumor, prior to administration of the cells or composition containing the cells. In some aspects, the subject is refractory or non-responsive to the other therapeutic agent. In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

In some embodiments, the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk for relapse, such as at a high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.

In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

The modified immune cells of the present invention can be administered to an animal, preferably a mammal, even more preferably a human, to treat a cancer. In addition, the cells of the present invention can be used for the treatment of any condition related to a cancer, especially a cell-mediated immune response against a tumor cell(s), where it is desirable to treat or alleviate the disease. The types of cancers to be treated with the modified cells or pharmaceutical compositions of the invention include, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Other exemplary cancers include but are not limited breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, and the like. The cancers may be non-solid tumors (such as hematological tumors) or solid tumors. Adult tumors/cancers and pediatric tumors/cancers are also included. In one embodiment, the cancer is a solid tumor or a hematological tumor. In one embodiment, the cancer is a carcinoma. In one embodiment, the cancer is a sarcoma. In one embodiment, the cancer is a leukemia. In one embodiment the cancer is a solid tumor.

Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).

Carcinomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.

Sarcomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

In certain exemplary embodiments, the modified immune cells of the invention are used to treat a myeloma, or a condition related to myeloma. Examples of myeloma or conditions related thereto include, without limitation, light chain myeloma, non-secretory myeloma, monoclonal gamopathy of undertermined significance (MGUS), plasmacytoma (e.g., solitary, multiple solitary, extramedullary plasmacytoma), amyloidosis, and multiple myeloma. In one embodiment, a method of the present disclosure is used to treat multiple myeloma. In one embodiment, a method of the present disclosure is used to treat refractory myeloma. In one embodiment, a method of the present disclosure is used to treat relapsed myeloma.

In certain exemplary embodiments, the modified immune cells of the invention are used to treat a melanoma, or a condition related to melanoma. Examples of melanoma or conditions related thereto include, without limitation, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma, amelanotic melanoma, or melanoma of the skin (e.g., cutaneous, eye, vulva, vagina, rectum melanoma). In one embodiment, a method of the present disclosure is used to treat cutaneous melanoma. In one embodiment, a method of the present disclosure is used to treat refractory melanoma. In one embodiment, a method of the present disclosure is used to treat relapsed melanoma. In yet other exemplary embodiments, the modified immune cells of the invention are used to treat a sarcoma, or a condition related to sarcoma. Examples of sarcoma or conditions related thereto include, without limitation, angiosarcoma, chondrosarcoma, Ewing’s sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, and synovial sarcoma. In one embodiment, a method of the present disclosure is used to treat synovial sarcoma. In one embodiment, a method of the present disclosure is used to treat liposarcoma such as myxoid/round cell liposarcoma, differentiated/dedifferentiated liposarcoma, and pleomorphic liposarcoma. In one embodiment, a method of the present disclosure is used to treat myxoid/round cell liposarcoma. In one embodiment, a method of the present disclosure is used to treat a refractory sarcoma. In one embodiment, a method of the present disclosure is used to treat a relapsed sarcoma.

The cells of the invention to be administered may be autologous, with respect to the subject undergoing therapy.

The administration of the cells of the invention may be carried out in any convenient manner known to those of skill in the art. The cells of the present invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In other instances, the cells of the invention are injected directly into a site of inflammation in the subject, a local disease site in the subject, alymph node, an organ, a tumor, and the like.

In certain embodiments, the cells, or individual populations of sub-types of cells, are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges.

In some embodiments, the dose of total cells and/or dose of individual sub-populations of cells is within a range of between at or about IxlO⁵ cells/kg to about IxlO¹¹ cells/kg 10⁴ and at or about 10¹¹ cells/kilograms (kg) body weight, such as between 10⁵ and 10⁶ cells / kg body weight, for example, at or about 1 x 10⁵ cells/kg, 1.5 x 10⁵ cells/kg, 2 x 10⁵ cells/kg, or 1 x 10⁶ cells/kg body weight. In other exemplary embodiments, a suitable dosage range of modified cells for use in a method of the present disclosure includes, without limitation, from about IxlO⁵ cells/kg to about IxlO⁶ cells/kg, from about IxlO⁶ cells/kg to about IxlO⁷ cells/kg, from about IxlO⁷ cells/kg about IxlO⁸ cells/kg, from about IxlO⁸ cells/kg about IxlO⁹ cells/kg, from about IxlO⁹ cells/kg about IxlO¹⁰ cells/kg, from about IxlO¹⁰ cells/kg about IxlO¹¹ cells/kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO⁸ cells/kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO⁷ cells/kg. In other embodiments, a suitable dosage is from about IxlO⁷ total cells to about 5xl0⁷ total cells. In some embodiments, a suitable dosage is from about IxlO⁸ total cells to about 5xl0⁸ total cells. In some embodiments, a suitable dosage is from about 1.4xl0⁷ total cells to about l.lxlO⁹ total cells. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about 7x10⁹ total cells.

In some embodiments, a dose of modified cells is administered to a subject in need thereof, in a single dose or multiple doses. In some embodiments, a dose of modified cells is administered in multiple doses, e.g., once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof by rapid intravenous infusion.

For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.

In some embodiments, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents includes a cytokine, such as IL-2, for example, to enhance persistence. In some embodiments, the methods comprise administration of a chemotherapeutic agent.

Following administration of the cells, the biological activity of the engineered cell populations in some embodiments is measured, e.g., by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying expression and/or secretion of one or more cytokines, such as CD 107a, IFNy, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.

In certain embodiments, the subject is provided a secondary or alternative treatment. Secondary/alternative treatments include but are not limited to chemotherapy, radiation, surgery, and medications.

In some embodiments, the subject can be administered a conditioning therapy, such as a lymphodepletion step, prior to adoptive cell therapy. In some embodiments, the conditioning therapy comprises administering an effective amount of cyclophosphamide to the subject. In some embodiments, the conditioning therapy comprises administering an effective amount of fludarabine to the subject. In preferred embodiments, the conditioning therapy comprises administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject. Administration of a conditioning therapy prior to adoptive cell therapy may increase the efficacy of the adoptive cell therapy. Methods of conditioning patients for T cell therapy are described in U.S. Patent No. 9,855,298, which is incorporated herein by reference in its entirety.

E. Chimeric Antigen Receptors

The present invention provides compositions and methods for cell-based immunotherapies. In certain embodiments, the cell-based immunotherapy comprises a CD33 knock-in HSPC-derived immune cell comprising a chimeric antigen receptor (CAR) (c.g., CAR macrophages, CAR NK cells). Thus, in some embodiments, the HSPC-derived immune cell has been genetically modified to express the CAR. CARs of the present invention comprise an antigen binding domain, a transmembrane domain, and an intracellular domain.

The antigen binding domain may be operably linked to another domain of the CAR, such as the transmembrane domain or the intracellular domain, both described elsewhere herein, for expression in the cell. In one embodiment, a first nucleic acid sequence encoding the antigen binding domain is operably linked to a second nucleic acid encoding a transmembrane domain, and further operably linked to a third a nucleic acid sequence encoding an intracellular domain.

The antigen binding domains described herein can be combined with any of the transmembrane domains described herein, any of the intracellular domains or cytoplasmic domains described herein, or any of the other domains described herein that may be included in a CAR of the present invention. A subject CAR of the present invention may also include a hinge domain as described herein. A subject CAR of the present invention may also include a spacer domain as described herein. In some embodiments, each of the antigen binding domain, transmembrane domain, and intracellular domain is separated by a linker.

Antigen Binding Domain

The antigen binding domain of a CAR is an extracellular region of the CAR for binding to a specific target antigen including proteins, carbohydrates, and glycolipids. In some embodiments, the CAR comprises affinity to a target antigen on a target cell. The target antigen may include any type of protein, or epitope thereof, associated with the target cell. For example, the CAR may comprise affinity to a target antigen on a target cell that indicates a particular disease state of the target cell.

In one embodiment, the target cell antigen is a tumor associated antigen (TAA). Examples of tumor associated antigens (TAAs), include but are not limited to, differentiation antigens such as MART-l/MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP- 180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG- 72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43- 9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS. In a preferred embodiment, the antigen binding domain of the CAR targets an antigen that includes but is not limited to CD19, CD20, CD22, ROR1, Mesothelin, CD33/IL3Ra, c-Met, PSMA, PSCA, Glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, and the like.

Depending on the desired antigen to be targeted, the CAR of the invention can be engineered to include the appropriate antigen binding domain that is specific to the desired antigen target. For example, if CD 19 is the desired antigen that is to be targeted, an antibody for CD 19 can be used as the antigen bind moiety for incorporation into the CAR of the invention. This should not be construed as limiting in any way, as a CAR having affinity for any target antigen is suitable for use in a composition or method of the present invention.

As described herein, a CAR of the present disclosure having affinity for a specific target antigen on a target cell may comprise a target-specific binding domain. In some embodiments, the target-specific binding domain is a murine target-specific binding domain, e.g., the target- specific binding domain is of murine origin. In some embodiments, the target-specific binding domain is a human target-specific binding domain, e.g., the target-specific binding domain is of human origin. For example, a CAR of the present disclosure having affinity for HER-2 on a target cell may comprise a HER-2 binding domain.

In some embodiments, a CAR of the present disclosure may have affinity for one or more target antigens on one or more target cells. In some embodiments, a CAR may have affinity for one or more target antigens on a target cell. In such embodiments, the CAR is a bispecific CAR, or a multispecific CAR. In some embodiments, the CAR comprises one or more target-specific binding domains that confer affinity for one or more target antigens. In some embodiments, the CAR comprises one or more target-specific binding domains that confer affinity for the same target antigen. For example, a CAR comprising one or more target-specific binding domains having affinity for the same target antigen could bind distinct epitopes of the target antigen. When a plurality of target-specific binding domains is present in a CAR, the binding domains may be arranged in tandem and may be separated by linker peptides. For example, in a CAR comprising two target-specific binding domains, the binding domains are connected to each other covalently on a single polypeptide chain, through an oligo- or polypeptide linker, an Fc hinge region, or a membrane hinge region.

In some embodiments, the antigen binding domain is selected from the group consisting of an antibody, an antigen binding fragment (Fab), and a single-chain variable fragment (scFv). For example, a CD19 binding domain of the present invention can be selected from the group consisting of a CD19-specific antibody, a CD19-specific Fab, and a CD19-specific scFv. In one embodiment, a CD 19 binding domain is a CD19-specific antibody. In one embodiment, a CD 19 binding domain is a CD19-specific Fab. In one embodiment, a CD19 binding domain is a CD19- specific scFv.

The antigen binding domain can include any domain that binds to the antigen and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, and any fragment thereof In some embodiments, the antigen binding domain portion comprises a mammalian antibody or a fragment thereof. The choice of antigen binding domain may depend upon the type and number of antigens that are present on the surface of a target cell. As used herein, the term “single-chain variable fragment” or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH: : VL heterodimer. The heavy (VH) and light chains (VL) are either joined directly or joined by a peptide-encoding linker, which connects the N- terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N- terminus of the VL. In some embodiments, the antigen binding domain (e.g., CD19 binding domain) comprises an scFv having the configuration from N-terminus to C-terminus, VH - linker - VL. In some embodiments, the antigen binding domain comprises an scFv having the configuration from N-terminus to C-terminus, VL - linker - VH. Those of skill in the art would be able to select the appropriate configuration for use in the present invention.

The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6): 1910-1917 (2008) and WO 2014/087010, the contents of which are hereby incorporated by reference in their entireties. Various linker sequences are known in the art, including, without limitation, glycine serine (GS) linkers such as (GS)n, (GSGGS)n (SEQ ID NO: 357), (GGGS)n (SEQ ID NO: 358), and (GGGGS)_n (SEQ ID NO: 359), where n represents an integer of at least 1. Exemplary linker sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO: 360), GGSGG (SEQ ID NO: 361), GSGSG (SEQ ID NO: 362), GSGGG (SEQ ID NO: 363), GGGSG (SEQ ID NO: 364), GSSSG (SEQ ID NO: 365), GGGGS (SEQ ID NO: 366), GGGGSGGGGSGGGGS (SEQ ID NO: 367) and the like. Those of skill in the art would be able to select the appropriate linker sequence for use in the present invention. In one embodiment, an antigen binding domain of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL is separated by the linker sequence having the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 368), which may be encoded by the nucleic acid sequence GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT (SEQ ID NO: 369).

Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VL-encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife eta., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3): 173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40). Agonistic scFvs having stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71 ; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

As used herein, “Fab” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two Fab fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).

As used herein, “F(ab')2” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab’) (bivalent) regions, wherein each (ab') region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S — S bond for binding an antigen and where the remaining H chain portions are linked together. A “F(ab')2” fragment can be split into two individual Fab' fragments.

In some embodiments, the antigen binding domain may be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen binding domain of the CAR may comprise a human antibody or a fragment thereof. In some embodiments, the antigen binding domain may be derived from a different species in which the CAR will ultimately be used. For example, for use in humans, the antigen binding domain of the CAR may comprise a murine antibody or a fragment thereof.

Transmembrane Domain

CARs of the present invention may comprise a transmembrane domain that connects the antigen binding domain of the CAR to the intracellular domain of the CAR. The transmembrane domain of a subject CAR is a region that is capable of spanning the plasma membrane of a cell (e g., an immune cell or precursor thereof). The transmembrane domain is for insertion into a cell membrane, e.g., a eukaryotic cell membrane. In some embodiments, the transmembrane domain is interposed between the antigen binding domain and the intracellular domain of a CAR.

In some embodiments, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some embodiments, the transmembrane domain can be selected or modified by one or more amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, to minimize interactions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein, e.g., a Type I transmembrane protein. Where the source is synthetic, the transmembrane domain may be any artificial sequence that facilitates insertion of the CAR into a cell membrane, e.g., an artificial hydrophobic sequence. Examples of the transmembrane domain of particular use in this invention include, without limitation, transmembrane domains derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

The transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the intracellular domains described herein, or any of the other domains described herein that may be included in a subject CAR.

In some embodiments, the transmembrane domain further comprises a hinge region. A subject CAR of the present invention may also include a hinge region. The hinge region of the CAR is a hydrophilic region which is located between the antigen binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding for the CAR. The hinge region is an optional component for the CAR. The hinge region may include a domain selected from Fc fragments of antibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial hinge sequences or combinations thereof. Examples of hinge regions include, without limitation, a CD8a hinge, artificial hinges made of polypeptides which may be as small as, three glycines (Gly), as well as CHI and CH3 domains of IgGs (such as human IgG4).

In some embodiments, a subject CAR of the present disclosure includes a hinge region that connects the antigen binding domain with the transmembrane domain, which, in turn, connects to the intracellular domain. The hinge region is preferably capable of supporting the antigen binding domain to recognize and bind to the target antigen on the target cells (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, the hinge region is a flexible domain, thus allowing the antigen binding domain to have a structure to optimally recognize the specific structure and density of the target antigens on a cell such as tumor cell (Hudecek et al., supra). The flexibility of the hinge region permits the hinge region to adopt many different conformations.

In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e g., a CD8-derived hinge region).

The hinge region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa. In some embodiments, the hinge region can have a length of greater than 5 aa, greater than 10 aa, greater than 15 aa, greater than 20 aa, greater than 25 aa, greater than 30 aa, greater than 35 aa, greater than 40 aa, greater than 45 aa, greater than 50 aa, greater than 55 aa, or more.

Suitable hinge regions can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitable hinge regions can have a length of greater than 20 amino acids (e.g., 30, 40, 50, 60 or more amino acids).

For example, hinge regions include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)_n, (GSGGS)n (SEQ ID NO: 357) and (GGGS)_n (SEQ ID NO: 358), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). Exemplary hinge regions can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 360), GGSGG (SEQ ID NO: 361), GSGSG (SEQ ID NO: 362), GSGGG (SEQ ID NO: 363), GGGSG (SEQ ID NO: 364), GSSSG (SEQ ID NO: 365), and the like.

In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al., Proc. Natl. Acad. Sei. USA (1990) 87(1): 162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4): 1779-1789. The hinge region can comprise an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge region. In one embodiment, the hinge region can include one or more amino acid substitutions and/or insertions and/or deletions compared to a wild-type (naturally- occurring) hinge region. For example, His229 of human IgGl hinge can be substituted with Tyr; see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897. In one embodiment, the hinge region can comprise an amino acid sequence derived from human CD8, or a variant thereof.

Intracellular Signaling Domain

A subject CAR of the present invention also includes an intracellular signaling domain. The terms “intracellular signaling domain” and “intracellular domain” are used interchangeably herein. The intracellular signaling domain of the CAR is responsible for activation of at least one of the effector functions of the cell in which the CAR is expressed (e.g., immune cell). The intracellular signaling domain transduces the effector function signal and directs the cell (e.g., immune cell) to perform its specialized function, e.g., harming and/or destroying a target cell.

Examples of an intracellular domain for use in the invention include, but are not limited to, the cytoplasmic portion of a surface receptor, co- stimulatory molecule, and any molecule that acts in concert to initiate signal transduction in the T cell, as well as any derivative or variant of these elements and any synthetic sequence that has the same functional capability. Examples of the intracellular signaling domain include, without limitation, the chain of the T cell receptor complex or any of its homologs, e.g., r| chain, FcsRIy and P chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (A, 8 and a), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain may be human CD3 zeta chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, an immunoreceptor tyrosine-based activation motif (IT AM) bearing cytoplasmic receptors, and combinations thereof.

In one embodiment, the intracellular signaling domain of the CAR includes any portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD2, CD3, CD8, CD27, CD28, ICOS, 4-1BB, PD-1, any derivative or variant thereof, any synthetic sequence thereof that has the same functional capability, and any combination thereof.

Other examples of the intracellular domain include a fragment or domain from one or more molecules or receptors including, but not limited to, TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), 0X9, 0X40, CD30, CD40, PD-1, ICOS, a KIR family protein, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD l id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CDlib, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD 18, LFA- 1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other co-stimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a co-stimulatory molecule that has the same functional capability, and any combination thereof. Additional examples of intracellular domains include, without limitation, intracellular signaling domains of several types of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, intracellular signaling domains may include signaling domains used by NK and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195) such as signaling domains of NKp30 (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP 12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP 10, and CD3z.

Intracellular signaling domains suitable for use in a subject CAR of the present invention include any desired signaling domain that provides a distinct and detectable signal (e g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation of the CAR (i.e., activated by antigen and dimerizing agent). In some embodiments, the intracellular signaling domain includes at least one (e.g., one, two, three, four, five, six, etc.) IT AM motifs as described below. In some embodiments, the intracellular signaling domain includes DAP10/CD28 type signaling chains. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane bound CAR, but is instead diffused in the cytoplasm.

Intracellular signaling domains suitable for use in a subject CAR of the present invention include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, an IT AM motif is repeated twice in an intracellular signaling domain, where the first and second instances of the IT AM motif are separated from one another by 6 to 8 amino acids. In one embodiment, the intracellular signaling domain of a subject CAR comprises 3 IT AM motifs.

In some embodiments, intracellular signaling domains includes the signaling domains of human immunoglobulin receptors that contain immunoreceptor tyrosine based activation motifs (IT AMs) such as, but not limited to, FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, FcRL5 (see, e.g., Gillis et al., Front. Immunol. (2014) 5:254). A suitable intracellular signaling domain can be an IT AM motif-containing portion that is derived from a polypeptide that contains an IT AM motif. For example, a suitable intracellular signaling domain can be an IT AM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

In one embodiment, the intracellular signaling domain is derived from DAP 12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX- activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer activating receptor associated protein; killer-activating receptor-associated protein; etc ). In one embodiment, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon Rl-gamma; fcRgamma; fceRl gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); 0KT3, delta chain; T-cell receptor T3 delta chain; T-cell surface glycoprotein CD3 delta chain; etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T- cell surface antigen T3/Leu-4 epsilon chain, T-cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 gamma chain (also known as CD3G, T-cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T-cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc ). In one embodiment, the intracellular signaling domain is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig- alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; etc ). In one embodiment, an intracellular signaling domain suitable for use in an FN3 CAR of the present disclosure includes a DAP10/CD28 type signaling chain. In one embodiment, an intracellular signaling domain suitable for use in an FN3 CAR of the present disclosure includes a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain includes a cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the CAR includes a cytoplasmic signaling domain of human CD3 zeta.

While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

The intracellular signaling domains described herein can be combined with any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that may be included in the CAR.

The invention should be construed to include any CAR known in the art and/or disclosed herein. Exemplary CARs include, but are not limited to, those disclosed herein, those disclosed in US10357514B2, US10221245B2, US10603378B2, US8916381B1, US9394368B2, US20140050708A1, US9598489B2, US9365641B2, US20210079059A1, US9783591B2, WO2016028896A1, US9446105B2, WO2016014576A1, US20210284752 Al, WO2016014565A2, WO2016014535A1, and US9272002B2, and any other CAR generally disclosed in the art.

F. Pharmaceutical Compositions

Also provided are compositions comprising populations of CD33 knock-in HSPCs comprising a CAR, CD33 knock-in HSPC-derived immune cells comprising a CAR (e.g.. CAR macrophages), NKG2A knock-in HSPCs comprising a CAR, and NKG2A knock-in HSPC- derived immune cells comprising a CAR (e.g., CAR NK cells) for use in immunotherapy. The population of cells can be generated by any of the methods contemplated herein. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.

Also provided are compositions including the cells for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.

The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).

Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine. The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.

Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term "parenteral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyoi (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.

Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by fdtration through sterile filtration membranes.

The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.

Example 1 : Recruitment, persistence and phenotype of CD33 -engineered cells in the TME

CD33 is redundant for human hematopoiesis and myeloid function. NOD/SCID/fL Ry-/- (NSG) mice were humanized using human CD34+ HSPC from which CD33 was deleted using CRISPR-based NHEJ. Mice were then challenged with the human SK0V3 cancer cell line. Tumor growth kinetics and immune cell recruitment to the TME were not altered by the absence of CD33 (FIGs. 2A-2D).

To track engineered cells, immunodeficient mice were engrafted with control or CD33^mCherry normal donor human HSPCs. Non-viral integration of a fluorescent reporter protein, mCherry, into the CD33 locus, was successfully achieved generating CD33^mCherry HSPCs. Persistent integration was confirmed by flow cytometry (FIGs. 9A-9B), and targeted PCR of the CD33 integration site. Moreover, CD33^mCherry cells were capable of efficiently engrafting NSG mice and persisting longterm, demonstrating the feasibility of CD33 gene engineered cells to reconstitute hematopoiesis (FIGs. 9C-9D), while simultaneously confining expression of the introduced genetic cargo to the myeloid compartment.

Example 2:

To create a durable source of CAR-expressing myeloid cells, primary CD34+ enriched human HSPCs from G-CSF mobilized normal donors are electroporated with a CD33-specific guide RNA (gRNA)/Cas9 ribonucleoprotein complex (RNP) to initiate a site-specific doublestrand break and allow for homology-directed repair (HDR). Cells are also transduced with an HDR repair template (HDRT) containing a CAR that recognizes the tumor-associated antigen HER-2. Upon HDR, the HER-2 CAR is expressed in-frame by the endogenous CD33 promoter in place of native CD33 protein (FIG. 3).

To ensure that CD33^HER2CAR myeloid cells differentiate from the engineered CD34+ HSPC in vivo as shown in preliminary data using CD33^mCherry myeloid cells, NSG mice are conditioned with busulfan prior to i.v. injection of l-5xl0⁵ CD34+ HSPC. Human hematopoietic engraftment of edited cells is quantified in the peripheral blood monthly. At 12-16 weeks post- engraftment, the phenotype, number, and distribution of cells within the bone marrow and major organs compared to control unedited cells is examined by flow cytometry and by fluorescent microscopy. Experiments are conducted in mice additionally xenografted with a relevant gastric cancer cell line to quantify human hematopoietic recruitment to the TME (SNU-216 or NCI- N87).

The impact of the TME on the engineered cells is studied by comparing the phenotype and function of intra-tumoral CD33^mCherry or CD33^HER2CAR and comparing these with CD33^mCherry and CD33^HER2CAR in the marrow, spleen and other organs of tumor bearing and non-tumor bearing mice. Flow cytometry, immunofluorescent (IF) microscopy and single cell RNAseq (scRNAseq) are used to define the interactions of HSPC-derived immune cells with tumor. Since NCI-N87 bears the tumor antigen HER-2, expression of this antigen is deleted from the cancer models in these experiments, which allows the impact of the tumor on the CAR-expressing infiltrating HSPC-derived immune cells in isolation to be studied.

CD34 cells from three individual human donors are used to account for inter-individual differences in gene editing efficiency and hematopoietic engraftment. Each mouse experiment consists of a control group receiving mock edited cells (electroporation with Cas9 protein without gRNA), an mCherry KI group (used as a reporter for HDR), and the experimental CAR KO group. Five mice in each group, for a total of 90 mice are used.

Delivery of a HDR template encoding a reporter gene (mCherry) or a CAR has no impact on hematopoietic differentiation, trafficking or infiltration into the TME. Some myeloid cells that infiltrate the TME adopt an immunosuppressive phenotype resembling “M2” macrophages and/or myeloid-derived suppressor cells, as shown in FIG. 2C (bottom panel, CD206 expression).

Example 3: Antitumor activity of CD33CAR-engineered cells

Using cells derived from control or CD33^HER2CAR and CD33^{cldnl8 2CAR}HSPCs, which express CARs targeting the cancer antigens HER2 or claudin 18.2 respectively, in vitro killing and phagocytosis assays are conducted to test their direct antitumor activity against antigen positive and negative UGI cancer cell lines. The same UGI cancer xenograft models described in Example 1 is used to assess direct in vivo antitumor function by bioluminescence and survival.

To test the antitumor activity of CZ)33^{CAR HFR2} HSPCs, primary human CD33^CAR'^HER2 HSPCs are differentiated to macrophage, monocyte, and dendritic cell lineages in vitro using either cytokine polarization or tumor-conditioned media, and their ability to phagocytose HER-2 positive and negative cancer cell lines are assessed. Phagocytosis and tumor killing is compared to control and CD33 knock-out only cells at different effector to target ratios (FIG. 15). Flow cytometry is used in these assays to assess myeloid cell phenotype, activation marker expression, and cytokine production during co-culture with cancer cell lines. Anti-tumor activity of HSPC- derived engineered myeloid cells bearing other CARs that are relevant to esophageal and gastric malignancies, including CD33^CAR'^{claudin18 2}, CD33^CAR'^TAG72 and CD33^CAR'^CEA, are also tested. Phagocytosis, killing and cytokine production of PB monocyte-derived CAR macrophages are directly compared with HSPC-derived CAR macrophages.

To test the function of edited HSPCs in an in vivo tumor model, NSG mice are engrafted with either control, CD33 -deficient (mCherry-expressing), or CD33^CAR'^HER2 HSPCs followed by administration of a luciferase/GFP expressing, HER-2 positive cancer cell line (e.g. SKOV-3, SKBR-3, NCI-N87). Tumor growth via in vivo bioluminescence is measured as well as mouse survival. CD33^CAR‘^{claudin 18 2}, CD33^CAR'^TAG72 and CD33^CAR’^CEAand others are tested in the same system to determine the optimal CAR construct.

Head-to-head comparisons are conducted by engrafting cohorts of mice with cancer cells followed by treatment with one of the antigen-specific CAR-KI CD34 cells, with the readouts being (i) frequency of tumor rejection, (ii) longterm relapse-free survival, (iii) toxicity (weight loss, necropsy). Experiments are repeated using cells derived from three individual human donors, to account for inter-individual differences in gene editing efficiency and hematopoietic engraftment.

CAR-expressing macrophages exhibit similar in vitro antitumor function whether these cells are derived from PB monocytes or from CD34+ HSPC. However, since the transduction efficiency of PB monocyte-derived CAR macrophages approaches 100% using the requisite adenoviral vector and that of CD34 HSPC-derived CAR macrophages engineered using the described CRISPR HDR system is closer to 10%, cell numbers are normalized to account for this. CAR-expressing HSPC-derived cells traffic to the growing tumor and exert direct antitumor activity, leading to tumor rejection while in the control groups (including CD33mCherry) tumor growth continues unabated.

Example 4: Indirect immune activity of CD33^CAR modified cells

CD33^CAR'^HER2 engineered HSPCs transform the TME of tumor-bearing mice towards an activated immune phenotype. Using flow cytometry, fluorescent microscopy, and single-cell RNA sequencing, the persistence of CD33^CAR'^HER2 myeloid cells is determined, as well as changes in composition and cell surface phenotype of the human immune infiltrate, and immune correlates of tumor rejection. Using a combination of genetic reporters and fluorochrome- conjugated antibodies tumor cells and CAR-expressing myeloid cells are distinguished, as well as the CAR-negative immune infiltrate. Myeloid, T cell, and B cell recruitment/trafficking, receptor expression, and cytokine production are quantified within the TME in mice engrafted with CD33^HER2CAR or control CD33^mCherry.

In addition to assessing direct cytotoxic function, the ability of CD33^CAR cells to engage the adaptive immune system as antigen presenting cells is tested using gastric cancer cell lines expressing the tumor-associated antigen (TAA) NY-ESO1. It has been shown that anti-HER2 CAR-M efficiently present antigen to and co-stimulate antigen-specific CD8+ T cells: Anti- HER2 CAR-M from an HLA-A*0201 human donor efficiently phagocytosed the human cancer cell line SK0V3 (these cells express the tumor-associated antigen NYES01 as well as HER2 but are negative for HLA-A*0201, therefore cannot by themselves be recognized by anti-NYESOl TCR-expressing CD8+ T cells). CD8 T cells from the same HLA-A*0201 donor were then transduced to express an anti-NYESOl TCR. T cell activation, proliferation and cytokine production were found to be highest in the presence of CAR-M that served as antigen presenting cells.

CD34 cells and T cells are obtained from an HLA-A*0201 healthy donor and engineered to express the anti-HER2 CAR in the CD33 locus as described herein. T cells are transduced with an anti-NYESOl TCR. The NCI-N87 gastric cell line (HLA haplotype A*23:01, 01 :01) is engineered to express NYESO1 (an additional subline is co-transduced to express the requisite HLA-A*0201 class I molecule to be used as a positive control). T cell stimulation is assayed using flow cytometry (CFSE dilution, acute activation marker CD69 and intracellular interferon- y production). To demonstrate antigen presentation in vivo, anti-NYESO (or control nonspecific) CD8 T cells are injected i.v. into tumor-bearing mice engrafted with CD33^CAR'^HER2, CD33^mCherry or control CD34 cells.

CAR+ myeloid cells in the TME lead to the development of a broadly proinflammatory phenotype consisting of “Ml” polarization of surrounding myeloid cells and activation of T cells, as shown in patient biopsies from clinical trials. CAR-bearing macrophages differentiated from engineered CD34+ cells efficiently present and co-stimulate antigen-specific T cells in vitro, and antigen-specific T cells mediate a more profound and prompt rejection of tumor in the presence of CAR-expressing HLA-matched myeloid cells in the TME than control conditions.

Example 5: Knock-in of therapeutic cargo into the CD33 locus

A schema for knock-in of therapeutic cargo into the CD33 locus is shown in FIG. 3. A DNA double-strand break is initiated at a lineage-specific targeted locus using CRISPR/Cas or other means and a template for homology-directed repair (HDRT) is provided either non-virally (dsDNA, ssDNA) or virally (adeno-associated virus/ AAV, integrase-deficient lentivirus/IDLV). The HDRT includes 5’ and 3’ homology arms (LHA/RHA) specific for the targeted gene. After undergoing HDR, cells express the inserted construct specifically within the targeted cell lineage. Various gRNAs targeting CD33 were screened (FIG. 4, Table 1). M0LM14 cell lines were electroporated with no guide RNA, a control guide RNA targeting exon 2 of CD33 known to cause CD33 knockout (SEQ ID NO: 169), or a number of additional guide RNAs in both full- length and truncated forms (Table 1). CD33 expression was assessed 14 days after electroporation by flow cytometry. Cell viability and CD33 knockout efficiency was most efficient for the truncated guide 4 (SEQ ID NO: 239), which targets CD33 exon 1 (FIG. 4). Genomic DNA was isolated from the M0LM14 cells after 14 days and targeted amplicons of CD33 were generated by PCR. Tracking indels by decomposition (TIDE) analysis showed indel frequency in cells targeted by either full-length (SEQ ID NO: 199) or truncated (SEQ ID NO: 239) Cas9 guide RNA/RNP complex using guide 4 (FIG. 5).

Table 1 : CD33 Guides Tested

Table 2: CD33 Guides Designed

An HDR template was designed for knock-in of mCherry into exon 1 of CD33 targeted by guide RNA 4 (SEQ ID NO: 239) (FIG. 6). CD33 engineering led to persistent gene expression in vitro (FIGs. 7-8). Primary human normal-donor (ND) CD34+ hematopoietic stem/progenitor cells (HSPCs) were electroporated with Cas9/ribonuclear protein (RNP) complex targeting either CD33 (CD33ko) or an irrelevant site (CD38ko) with non-viral dsDNA HDRT encoding for the fluorescent reporter protein mCherry (SEQ ID NO: 150). Targeted integration of mCherry could be detected by flow cytometry in HSPC with on-target RNP (CD33) but not irrelevant (CD38ko) and was maintained >1 month during myeloid and macrophage differentiation.

Table 3: HDRT Sequences

Table 4: HDRT Amplification Primers

Table 5: Sequencing Primers

Primary normal donor CD34+ human HSPC were edited using Cas9 RNP containing either no guide RNA (NTC), CD33 guide RNA (33KO) (SEQ ID NO: 239), or an off-target guide RNA (38KO) specific for the unrelated gene CD38 (SEQ ID NO: 170) (FIG. 8). Indicated conditions were co-electroporated with a dsDNA HDRT encoding for mCherry (mC) (SEQ ID NO: 150). Cells were cultured in vitro, and genomic DNA was isolated after 7 days, followed by PCR amplification using primers targeting the CD33 locus beyond the 3’ homology arm/RHA (SEQ ID NO: 180) and the mCherry insert (SEQ ID NO: 185) and agarose gel electrophoresis. The box indicates amplicon of expected size representing on-target insertion, which was confirmed by gel purification, Sanger sequencing of the PCR product, and alignment with the expected insertion sequence (FIG. 8, right panel). Genomic detection of on-target CD33 HDR knock-in HSPC was demonstrated. In the alignment shown in FIG. 8, right panel, the top sequence in the alignment (designated nucleotides 1726-2293; “CD33 ENST”) is shown as: ACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCC ACCGGCGGCATGGACGAGCTGTACAAGGCTACTAACTTCAGCCTGCTGAAGCAGGC TGGCGACGTGGAGGAGAACCCTGGACCTGCTAGCTCCAGATAAGTCGACCATGGCC CAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTT CACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAAT GTATCTTATCATGTCTGGATCGGGAATCCTGTGGGCAGGTGAGTGGCTGTGGGGAGA GGGGTTGTCGGGCTGGGCCGAGCTGACCCTCGTTTCCCCACAGGGGCCCTGGCTATG GATCCAAATTTCTGGCTGCAAGTGCAGGAGTCAGTGACGGTACAGGAGGGTTTGTG CGTCCTCGTGCCCTGCACTTTCTTCCATCCCATACCCTACTACGACAAGAACTCCCCA GTTCATGGTTACTGGTTCCGGGAAGGAGCCATTATATCCAGGGACTCTCCAGTGGCC (SEQ ID NO: 347); and bottom starnd sequence in the alignment (“05.7_E01”) is shown as: TCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGAC GAGCTGTACAAGGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGCGACGTGGAGGA GAACCCTGGACCTGCTAGCTCCAGATAAGTCGACCATGGCCCAACTTGTTTATTGCA GCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTT TTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCT GGATCGGGAATCCTGTGGGCAGGTGAGTGGCTGTGGGGAGAGGGGTTGTCGGGCTG GGCCGAGCTGACCCTCGTTTCCCCACAGGGGTCCTGGCTATGGATCCAAATTTCTGG CTGCAAGTGCAGGAGTCAGTGACGGTACAGGAGGGTTTGTGCGTCCTCGTGCCCTGC ACTTTCTTCCATCCCATACCCTACTACGACAAGAACTCCCCAGTTCATGGTTACCKGT TCCGGAARGWAGSCRCAMKCG (SEQ ID NO: 348).

Engraftment of CD33 knock-in primary human HSPCs is shown in FIGs. 9A-9D. Control (Ctrl), CD33 knockout (CD33ko), or CD33-knockout/mCheriy knock-in (CD33mCherry) primary human normal donor (n.d.) CD34+ HSPCs were generated by Cas9/gRNA (SEQ ID NO: 239) RNP electroporation +/- a dsDNA HDRT encoding mCherry (SEQ ID NO: 150). mCherry knock-in was detected by flow cytometry after 4 days (FIG. 9A). NSG mice were engrafted with control, CD33ko, or CD33mCherry human HSPC after busulfan conditioning (FIG. 9B). mCherry expression was detected in peripheral blood monocytes for >16 weeks by flow cytometry. NSG mice engrafted with HSPC were challenged at 8 weeks with SKOV3 cancer cells i.v. (FIG. 9C). Tumor-bearing lung digests at harvest showed mCherry+ human immune infdtration of the TME by flow cytometry (gated on live/human CD45+/mouse CD45-). TME mCherry expression was restricted to HLA-DR+ APCs, which co-expressed the macrophage marker CD1 lb, but was not found in T (CD3+), B (CD19+) or NK (CD56+) cells (FIG. 9D). -30% of TME macrophages were mCherry+, reflecting -10% of the total human CD45+ infiltrate.

The CD33 knock-in strategy is effective in all HSPC subsets, including phenotypically immature hematopoietic stem cells (FIGs. 10A-10B). mCherry reporter was detected by flow cytometry within indicated human lineage-positive (Lin+) and Lin- cell populations in the bone marrow of NSG mice engrafted with control (Ctrl), CD33 knockout (CD33ko) or CD33 knock-in (CD33mCherry) human HPSCs (FIG. 10B). Cells were detected 16 weeks post-engraftment by gating on human CD45+/murine CD45- on harvested single-cell suspensions from isolated bone marrow.

CD33 engineered HSPCs maintained hematopoietic potency on secondary bone marrow engraftment (FIGs. 11 A-l IB). After 12 weeks, secondarily engrafted NSG mice were sacrificed and bone marrow harvested for flow cytometry. After gating on human CD45+/murine CD45- cells, mCherry was detected in both the total (FIG. 1 IB, top panel) and CD34+ (FIG. 35B, bottom panel) only in mice engrafted with CD33 knock-in cells.

Constructs used for CAR knock-in to the CD33 locus are shown in FIG. 12. Therapeutic chimeric antigen receptors (CARs) were knocked-in to the CD33 locus of primary HSPCs (FIG. 13). CD33 knock-in primary human normal donor (n.d.) CD34⁺ HSPCs were generated by CD33 Cas9/gRNA (SEQ ID NO: 239) RNP electroporation +/- a dsDNA HDRT encoding for a control HER2 CAR (SEQ ID NO: 151) (Ctrl ki, not antibody detectable), anti-CD33 CAR (CAR33/containing a G4S scFv linker) (SEQ ID NO: 148), anti-Her2 CAR (HER2CAR) containing a Myc Tag (5177 ki) (SEQ ID NO: 146), or HER2CAR containing a G4S scFv linker (5178 ki) (SEQ ID NO: 145). Five days after electroporation, surface expression of CAR was detected by antibody staining for the G4S linker or Myc tag. Stable genomic detection of on-target CD33 CAR knock-in was achieved (FIGs. 14A- 14D). Primary normal donor CD34+ human HSPCs were edited using a CD33 targeting Cas9 RNP with either mCherry (mC) (SEQ ID NO: 150) or Her2CAR (5178) (SEQ ID NO: 145) dsDNA HDRT. PCR amplification was performed using a primer targeting the CD33 locus beyond the 5’ homology arm/LHA (SEQ ID NO: 171) and a second primer specific for the Her2CAR insert for 5178 ki (SEQ ID NO: 173) (or the mCherry insert for mC ki; SEQ ID NO: 181) (FIG. 14A). The box indicates the amplicon of expected size detected by agarose gel electrophoresis representing on-target insertion of the 5178 Her2 CAR construct (SEQ ID NO: 145) (FIG. 14A). On-target insertion was confirmed by gel purification, Sanger sequencing of the PCR product, and alignment with the expected insertion sequence (FIG. 14B). In FIG. 14B, alignment, top strand sequence is: TCCGGCCCTGTAGTCCTTCCCCTCCACTCCCTTCCTCTTTTCTGCTCACACAGGAAGC CCTGGAAGCTGCTTCCTCAGACATGCCGCTGCTGCTACTGCTGCCACTGGGATCGGG TGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGCGACGTGGAGGAGAACCCTGGAC C (SEQ ID NO: 349); and bottom strand sequence is: ACCCGTTRTCCTTCCTCACTCCCTTCCTCTTTTCTGCTCACACAGGAAGCCCTGGAAG CTGCTTCCTCAGACATGCCGCTGCTGCTACTGCTGCCMCTGSKATCGGGTGCTACTA AMTTYWGCCTGCTGAAKCAGGCTGGCGACGTGGAGGAGAACCCTGGACC (SEQ ID NO: 350).

Her2CAR knock in (5178 ki) CD34+ HSPCs were sorted for CAR positivity by fluorescence activated cell sorting (FACS) and differentiated into CD 14+ macrophages for 60 days (FIG. 14C). CAR surface expression remained detectable by flow cytometry in -90% of sorted 5178 ki cells but was not detectable in mCherry knock-in (mC ki) macrophages. Genomic DNA was extracted from macrophages after 60 days and amplified using an unbiased 3 -primer PCR reaction to generate amplicons that included both wild-type/indel CD33 alleles and the Her2 CAR knockin (FIG. 14D). Over 60% of sequences in 5178 ki macrophages showed on- target CAR insertion (HDR) by Inference of Crispr Edits (ICE) analysis. Sequencing of control amplicons generated using the same primers from mC ki macrophages showed only wild-type alleles or indels but no HDR knock-in of CAR.

Peripheral blood monocytes expressing mCherry and anti-Her2 CAR expression were detected in peripheral blood monocytes by flow cytometry (FIG. 15A). Further, CD33 knock-in HSPC-derived CAR macrophages displayed functional antitumor activity (FIG. 15B). CD33 knockout/Her2 CAR knock-in cells were sorted by FACS and differentiated to macrophages for 21 days prior to incubation with the Her2+ cancer cell line SK0V3 expressing click beetle green luciferase for 48 hours. Normalized specific lysis was calculated as decline in luciferin fluorescence normalized to control unedited macrophages and was compared to cells with knock- in of a non-HER2 specific CAR (irrelevant CAR).

Example 6: Engraftment of CD33^^^^ primary human HSPCs in immunodeficient NSG mice.

Detection of peripheral blood monocytes expressing mCherry and Her2 CAR in NSG mice engrafted with control (Ctrl), CD33 knockout (CD33ko), CD33 knock-in mCherry (CD33^mCherry), and CD33 knock-in Her2 CAR (CD33^HER2CAR) human HPSCs was examined in NSG mice mice by flow cytometry. CD34+ HSPCs generated by Cas9/gRNA RNP electroporation +/- a dsDNA HDRT encoding mCherry or HER2 CAR were injected in NSG mice and cultured in MethoCult medium two weeks before collection of peripheral blood monocytes for analysis. FIG. 16A shows a protocol for the detection of mCherry and Her2 CAR where quantification of the percentage of peripheral blood monocytes expressing mCherry and Her2 CAR (FIGs. 16B-C) by flow cytometry. Control (Ctrl), CD33 knockout (CD33ko), CD33- knockout/mCherry knock-in (CD33^mCherry), or CD33 knockout/HER2 CAR knock in (CD33^{HER2 CAR}) primary human normal donor (n.d.) CD34+ HSPCs were generated by Cas9/gRNA RNP electroporation +/- a dsDNA HDRT encoding mCherry or HER2 CAR. NSG mice were engrafted with control, CD33ko, CD33^mChe,Ty, or human CD33^{HER2 CAR} human HSPC (FIG. 16A). Peripheral blood monocytes expressing mCherry and anti-Her2 CAR expression were detected in peripheral blood monocytes by flow cytometry (FIG. 16B-C) and a MethoCult assay was performed to determine the percentages of mCherry- and HER2 CAR-expressing cells within CFU-GM colonies (granulocyte/macrophage progenitors, identified by expression of CD 14) using flow cytometry (FIG. 16D)

As expected, mCherry and Her2 CAR was detected in the peripheral blood monocytes (FIG. 16B). The percentages of mCherry- and Her2 CAR-expressing monocytes was determined as shown in FIG. 16C. No expression of mCherry or anti-Her2 CAR was detected in the control or CD33ko cells. Example 7: Engraftment of myeloid cells leads to enhanced T cell infiltration

in the TME of NSGS mice.

Triple transgenic NSGS mice expressing human IL3, GM-CSF (CSF2) and SCF (KITLG) were used as a model to evaluate immune cell infiltration in the TME. NSGS mice are particularly useful for supporting the stable engraftment of myeloid lineages and regulatory T cell populations. NSGS mice engrafted with CD33^mCherry and human CD33^{HER2 CAR} were challenged with the Her2+ cancer cell line SK0V3 to generate uniform manifold approximation and projection (UMAP) plots of the engrafted of human CD33^mC11L'^rrv and human CD33^{HER2 CAR} HSPCs. FIG. 17A shows a protocol for this analysis. Single-cell RNA sequencing (scRNA seq) of tumors was used to generate the UMAP plots of the human CD33^mCherry and human CD33^HER2CAR HSPCS, which are colored by dataset and by cell types infiltrating the tumors (FIGs. 17B-17D).

Example 8: Engraftment of CD33^HER2CARi; myeloid cells in NSGS mice leads to enhanced CCL2 and fFNy-related chemokine expression

FIG. 18A shows a protocol for examining chemokine expression in peripheral blood monocytes following injection of NSGS mice with Her2+ SKOV cells and engrafted CD33^HER2CAR or CD33^mCherry CD34+ HSPCs. Peripheral blood monocytes were collected 4-11 weeks post-engraftment. FIGs. 18B-18E show expression levels of CCL2 (FIG. 18A); CXCL9, CXCL10, and CXCL11 (FIG. 18C); CCL17 and CCL20 (FIG. 18D); and CXCL5, CXCL1, and CXCL8 (FIG. 18E) in CD33^mChe,Ty and CD33^HER2CAR engrafted myeloid cells.

Significantly increased expression levels of CCL2 (FIG. 18B) and the IFNy-related CXCL9 and CXCL10 chemokines (FIG. 18C) were observed in the CD33^HER2CAR engrafted myeloid cells relative to the CD33^mCherry. Minor increases in CXCL11 (FIG. 18C) and CXCL8 (FIG. 18E) expression levels were observed, while no significant changes in expression levels were observed for CCL17 and CCL20 (FIG. 18D) or CXCL5 and CXCL1 (FIG. 18E).

To examine the effect of CD3(j or CD3y intracellular signaling domains in Her2 CAR on CCL2 and CXCL9 chemokine expression in CD33^HER2CAR engrafted myeloid cells, peripheral blood monocytes were collected following injection of NSGS mice with Her2+ SKOV cells and the engrafted CD33^mCheiTy, CD33^IIER2CARi;, or CD33^IIER2CARY CD34+ HSPCs. FIG. 19A shows a protocol for this analysis. The monocytes were evaluated for expression of CCL2 (FIG. 19B) or CXCL9 (FIG. 19C). The results of this analysis show that increased expression levels of CCL2 and CXCL9 appear to require CD3(^ signaling, since the increased expression levels were observed with engrafted myeloid cells containing an anti-HER2 CAR with a CD3(^ signaling domain, but not a CD3y signaling domain (FIGs. 19B and 19C, middle lanes)

Example 9: Time course of myeloid marker and CAR expression associated with peripheral blood engraftment in NSGS mice following challenge with Her2+ SKOV3 cells

To assess engineered HSPC engraftment in a tumor model, primary normal donor CD34+ human hematopoietic stem/progenitor cells (HSPCs) were electroporated on day -1 (D-l) with no guide RNA (EP only), or CD33 targeting guide RNA with HDRT knock-in of mCherry (CD33^mCherry) or Her2CAR (CD33^HER2z) prior to injection into busulfan-conditioned NSG-SGM3 (NSGS) mice on DO (FIG. 20A). The expression time course of CD14, CD3, and CD19 expression was examined following injection of engrafted CD33^HER2CAR or CD33^mCherry CD34+ HPSC cells in NSGS mice that were subsequently challenged with Her2+ SKOV3 cells. Peripheral blood engraftment of indicated cell types based on canonical markers was assessed by flow cytometry of peripheral blood at indicated timepoints (monocytes - CD14, B cells - CD19, T cells- CD3). The number of peripheral blood monocytes expressing CD14, CD3, and CD19 per pL as a function of time was determined. The results of this analysis demonstrated equivalent cell numbers for CD33 engineered versus control cells with no significant differences (FIGs. 20B-20C).

Additionally, the expression time course of mCherry and Her2 CAR expression was examined following injection of engrafted CD33^HER2CARor CD33^mCherry CD34+ HPSC cells in NSGS mice subsequently challenged with Her2+ SK0V3 cells. FIG. 21A shows an experimental protocol for examining stable engraftment of mCherry and anti-HER2 CAR . Primary normal donor CD34+ human hematopoietic stem/progenitor cells (HSPCs) were electroporated on day -1 (D-l) with no guide RNA (EP only), or CD33 targeting guide RNA with HDRT knock-in of mCherry (CD33mCherry) or Her2CAR (CD33HER2z) prior to injection into busulfan-conditioned NSG-SGM3 (NSGS) mice on DO. mCherry or HER2-CAR expression was assessed by flow cytometry of peripheral blood monocytes at indicated timepoints (FIGs. 21B-21C). FIGs. 21B-21C show the number of peripheral blood monocytes expressing CD33^inCherry or CD33^HER2CAR per pl. as a function of time, respectively, and show that equivalent cell numbers of CD33 engineered versus control cells were obtained with no significant differences.

Example 10: Antitumor activity of anti-Her2 CAR knock-in HSCs in vivo

Using cells derived from control (Ctrl), CD33-deficient mCherry-expressing, or CD3- defi cient Her2 CAR-expressing HSPCs, the antitumor activity of the edited HSPCs was examined in an in vivo tumor model. FIG. 22 A shows a protocol for examining antitumor activity of CD33^{HER2 CAR} CD34+ HSPCs in terms of tumor growth (FIG. 22B) and survival of NSGS mice as a function of time. NSGS mice engrafted in Fig 20-21 were challenged after 4 weeks of CD34 engraftment with Her2+ SKOV3 ovarian cancer cells expressing GFP and clickbeetle green (CBG) luciferase. Tumor growth was monitored over time by in vivo bioluminescence imaging and survival proportion. Bioluminescence showed a significant decrease in tumor burden at late timepoints in CD33^HER2CARz engrafted mice compared to CD33^mCherry engrafted animals (FIG. 22B), with a non-significant trend (p=0.097) toward improved survival in CD33^HER2CARz engrafted mice by log rank test (FIG. 22C).

Example 11 : Knock-in of therapeutic cargo into the NKG2A/KLRC1 locus

A schema for therapeutic cargo knock-in to the NKG2A/KLRC1 locus is shown in FIG. 23. A DNA double-strand break is initiated at a lineage-specific targeted locus using CRISPR/Cas or other means and a template for homology-directed repair (HDRT) is provided either nonvirally (dsDNA,ssDNA) or virally (adeno-associated virus/AAV, integrase-deficient lentivirus/IDLV). HDR template design for knock-in of CAR33 and tEGFR into exon 2 of NKG2A/KLRC1 is shown in FIGs. 26A-B, respectively. FIG. 26C shows additional details accompanying FIG. 26A. The HDRT includes 5’ and 3’ homology arms (LHA/RHA) specific for the targeted gene. After undergoing HDR cells express the inserted construct specifically within the targeted cell lineage (NK cells).

Table d: KLRC1 gRNAs

Table 7: KLRC1 HDRTs

Table 8: HDRT Amplification Primers

EGFRt knock-in to NKG2A/KLRC1 in primary human NK cells was demonstrated herein (FIGs. 24A-24B). Primary human normal donor Natural Killer (NK) cells were electroporated with Cas9/sgRNA (SEQ ID NO: 186) RNP targeted KLRC1 with/without a double-stranded DNA HDR template (HDRT) encoding a truncated non-signaling epidermal growth factor receptor (EGFRt) (SEQ ID NO: 191) after culture for 16 hours with IL-12, 15, and 18 followed by maintenance and expansion with IL-2. Flow cytometry plots showed successful tEGFR detection and NKG2A knockout after 7 days of in vitro culture in EGFR knock-in (KLRClEGFRt) (SEQ ID NO: 191) but not mock electroporated (Ctrl) or NKG2A knockout- only (KLRC1 ko) controls (FIGs. 24A-24B).

EGFRt knock-in to NKG2A/KLRC 1 in primary human HSPCs was followed by NK cell differentiation (FIG. 25). Primary normal donor human HSPCs were electroporated with Cas9/sgRNA RNP targeting KLRC1 with/without a double- stranded DNA HDR template (HDRT) (SEQ ID NO: 191) encoding a truncated non-signaling epidermal growth factor receptor (EGFRt) followed by culture for 18 days with StemSpam NK cell generation kit (StemCell Technologies). EGFRt knock-in was detected by flow cytometry in knock-in (KLRClEGFRt) but not mock electroporation control (Ctrl).

An HDR template design for knock-in of CAR33 and tEGFR into exon 2 of NKG2A/KLRC1 of primary HSPCs is shown in FIGs. 26A-B, respectively. FIG. 26C shows additional details accompanying FIG. 26A. Primary normal donor CD56+ human NK cells were edited using a KLRC1 targeting Cas9 RNP with CAR33 dsDNA through electroporation +/- a dsDNA HDRT encoding for an anti-CD33 CAR (CAR33/containing a G4S scFv linker). 7 days after electroporation, surface expression of CAR was detected by flow cytometry using an antibody specific for the G4S linker (FIG. 27A). Sanger sequencing of PCR amplification products was performed using one primer targeting the KLRC1 locus beyond the 5’ homology arm/LHA and a second primer specific for the CAR33 insert (EFl A promotor). FIG. 27B shows alignment with the expected insertion sequence (FIG. 27B).

NSG mice engrafted with control (Ctrl) or KLRC1-CRISPR knockout (NKG2Ako) human HSPCs showed NKG2Ako persistence (right) and equivalent blood engraftment and splenic infiltrations (left) (FIG. 28). Antitumor activity of NKG2A knock-in CAR33 and EGFRt NK cells and mock controls were examined by co-incubation with the CD33+ cancer cell line M0LM14 expressing click beetle green luciferase for 24 hours at different effector to target ratios (1 : 1, 1 :4 and 1 :8). Normalized specific lysis was calculated as decline in luciferin fluorescence normalized to control unedited macrophages and compared to cells with knock-in of a non-HER2 specific CAR (irrelevant CAR). As shown in FIG. 29, NKG2A knock-in CAR33 and EGFRt NK cells displayed displayed functional antitumor activity.

Enumerated Embodiments

The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance.

Embodiment 1 provides a modified hematopoietic stem/progenitor cell (HSPC) comprising a modification in the endogenous CD33 locus, wherein an exogenous nucleic acid has been inserted into the CD33 locus.

Embodiment 2 provides the HSPC of embodiment 1, wherein the exogenous nucleic acid encodes a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain.

Embodiment 3 provides the HSPC of embodiment 2, wherein the antigen binding domain binds CD33 or HER-2.

Embodiment 4 provides the HSPC of embodiment 1, wherein the exogenous nucleic acid encodes IL-12.

Embodiment 5 provides the HSPC of any preceding embodiment, wherein the HSPC differentiates into an immune cell.

Embodiment 6 provides the HSPC of embodiment 5, wherein the immune cell is a monocyte or macrophage.

Embodiment 7 provides a modified hematopoietic stem/progenitor cell (HSPC) comprising a modification in the endogenous NKG2A locus, wherein an exogenous nucleic acid has been inserted into the NKG2A locus.

Embodiment 8 provides the HSPC of embodiment 7, wherein the exogenous nucleic acid encodes a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain.

Embodiment 9 provides the HSPC of embodiment 8, wherein the antigen binding domain binds CD33 or HER-2.

Embodiment 10 provides the HSPC of embodiment 7, wherein the exogenous nucleic acid encodes IL-12. Embodiment 1 1 provides the HSPC of any of embodiments 7-10, wherein the HSPC differentiates into an immune cell.

Embodiment 12 provides the HSPC of embodiment 5, wherein the immune cell is a Natural Killer (NK) cell.

Embodiment 13 provides a method of generating a modified immune cell, the method comprising: introducing an exogenous nucleic acid into the CD33 locus of a hematopoietic stem/progenitor cell (HSPC), and allowing the HSPC to differentiate into an immune cell.

Embodiment 14 provides the method of embodiment 13, wherein the exogenous nucleic acid is introduced via a CRISPR/Cas system.

Embodiment 15 provides the method of embodiment 14, wherein the CRISPR/Cas system comprises Cas9, a guide RNA (gRNA) that targets the CD33 locus, and a template for homology-directed repair (HDRT) comprising a nucleotide sequence that encodes the exogenous nucleic acid.

Embodiment 16 provides the method of generating a modified immune cell comprising a chimeric antigen receptor (CAR), the method comprising: introducing an exogenous nucleic acid encoding the CAR into the CD33 locus of a hematopoietic stem/progenitor cell (HSPC), and allowing the HSPC to differentiate into an immune cell.

Embodiment 17 provides the method of embodiment 16, wherein the exogenous nucleic acid is introduced via a CRISPR/Cas system.

Embodiment 18 provides the method of embodiment 17, wherein the CRISPR/Cas system comprises Cas9, a guide RNA (gRNA) that targets the CD33 locus, and a template for homology-directed repair (HDRT) comprising a nucleotide sequence that encodes the exogenous CAR.

Embodiment 19 provides the method of any of embodiments 13-18, wherein the immune cell is a monocyte or macrophage.

Embodiment 20 provides a method of generating a modified immune cell, the method comprising: introducing an exogenous nucleic acid into the NKG2A locus of a hematopoietic stem/progenitor cell (HSPC), and allowing the HSPC to differentiate into an immune cell.

Embodiment 21 provides the method of embodiment 20, wherein the exogenous nucleic acid is introduced via a CRISPR/Cas system. Embodiment 22 provides the method of embodiment 21, wherein the CRISPR/Cas system comprises Cas9, a guide RNA (gRNA) that targets the NKG2A locus, and a template for homology-directed repair (HDRT) comprising a nucleotide sequence that encodes the exogenous nucleic acid.

Embodiment 23 provides a method of generating a modified immune cell comprising a chimeric antigen receptor (CAR), the method comprising: introducing an exogenous nucleic acid encoding the CAR into the NKG2A locus of a hematopoietic stem/progenitor cell (HSPC), and allowing the HSPC to differentiate into an immune cell.

Embodiment 24 provides the method of claim 23, wherein the exogenous nucleic acid is introduced via a CRISPR/Cas system.

Embodiment 25 provides the method of claim 24, wherein the CRISPR/Cas system comprises Cas9, a guide RNA (gRNA) that targets the NKG2A locus, and a template for homology-directed repair (HDRT) comprising a nucleotide sequence that encodes the exogenous CAR.

Embodiment 26 provides the method of any of embodiments 23-25, wherein the immune cell is aNK cell.

Embodiment 27 provides a pharmaceutical composition comprising the HSPC or population thereof of any of claims 1-12, or the immune cell or population thereof generated by any of embodiments 13-26.

Embodiment 28 provides a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject, the pharmaceutical composition of embodiment 27.

Embodiment 29 provides the method of embodiment 28, wherein the disease is cancer.

Embodiment 30 provides the method of embodiment 28, wherein the immune cells are capable of long-term persistence in vivo.

Embodiment 31 provides the method of embodiment 28, wherein administering the pharmaceutical composition alters the tumor microenvironment (TME).

The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed:

1. A modified hematopoietic stem/progenitor cell (HSPC) comprising a modification in the endogenous CD33 locus, wherein an exogenous nucleic acid has been inserted into the CD33 locus.

2. The HSPC of claim 1, wherein the exogenous nucleic acid encodes a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain.

3. The HSPC of claim 2, wherein the antigen binding domain binds CD33 or HER-2.

4. The HSPC of claim 1, wherein the exogenous nucleic acid encodes IL-12.

5. The HSPC of any preceding claim, wherein the HSPC differentiates into an immune cell.

6. The HSPC of claim 5, wherein the immune cell is a monocyte or macrophage.

7. A modified hematopoietic stem/progenitor cell (HSPC) comprising a modification in the endogenous NKG2A locus, wherein an exogenous nucleic acid has been inserted into the NKG2A locus.

8. The HSPC of claim 7, wherein the exogenous nucleic acid encodes a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain.

9. The HSPC of claim 8, wherein the antigen binding domain binds CD33 or HER-2.

10. The HSPC of claim 7, wherein the exogenous nucleic acid encodes IL-12.

11. The HSPC of any of claims 7-10, wherein the HSPC differentiates into an immune cell.

12. The HSPC of claim 5, wherein the immune cell is a Natural Killer (NK) cell.

13. A method of generating a modified immune cell, the method comprising: introducing an exogenous nucleic acid into the CD33 locus of a hematopoietic stem/progenitor cell (HSPC), and allowing the HSPC to differentiate into an immune cell.

14. The method of claim 13, wherein the exogenous nucleic acid is introduced via a CRISPR/Cas system.

15. The method of claim 13, wherein the CRISPR/Cas system comprises Cas9, a guide RNA (gRNA) that targets the CD33 locus, and a template for homology-directed repair (HDRT) comprising a nucleotide sequence that encodes the exogenous nucleic acid.

16. A method of generating a modified immune cell comprising a chimeric antigen receptor (CAR), the method comprising: introducing an exogenous nucleic acid encoding the CAR into the CD33 locus of a hematopoietic stem/progenitor cell (HSPC), and allowing the HSPC to differentiate into an immune cell.

17. The method of claim 16, wherein the exogenous nucleic acid is introduced via a CRISPR/Cas system.

18. The method of claim 17, wherein the CRISPR/Cas system comprises Cas9, a guide RNA (gRNA) that targets the CD33 locus, and a template for homology-directed repair (HDRT) comprising a nucleotide sequence that encodes the exogenous CAR.

19. The method of any of claims 13-18, wherein the immune cell is a monocyte or macrophage.

20. A method of generating a modified immune cell, the method comprising: introducing an exogenous nucleic acid into the NKG2A locus of a hematopoietic stem/progenitor cell (HSPC), and allowing the HSPC to differentiate into an immune cell.

21. The method of claim 20, wherein the exogenous nucleic acid is introduced via a CRISPR/Cas system.

22. The method of claim 21, wherein the CRISPR/Cas system comprises Cas9, a guide RNA (gRNA) that targets the NKG2A locus, and a template for homology-directed repair (HDRT) comprising a nucleotide sequence that encodes the exogenous nucleic acid.

23. A method of generating a modified immune cell comprising a chimeric antigen receptor (CAR), the method comprising: introducing an exogenous nucleic acid encoding the CAR into the NKG2A locus of a hematopoietic stem/progenitor cell (HSPC), and allowing the HSPC to differentiate into an immune cell.

24. The method of claim 23, wherein the exogenous nucleic acid is introduced via a CRISPR/Cas system.

25. The method of claim 24, wherein the CRISPR/Cas system comprises Cas9, a guide RNA (gRNA) that targets the NKG2A locus, and a template for homology-directed repair (HDRT) comprising a nucleotide sequence that encodes the exogenous CAR.

26. The method of any of claims 23-25, wherein the immune cell is a NK cell.

27. A pharmaceutical composition comprising the HSPC or population thereof of any of claims 1-12, or the immune cell or population thereof generated by any of claims 13-26.

28. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject, the pharmaceutical composition of claim 27.

29. The method of claim 28, wherein the disease is cancer.

30. The method of claim 28, wherein the immune cells are capable of long-term persistence in vivo.

31. The method of claim 28, wherein administering the pharmaceutical composition alters the tumor microenvironment (TME).